Anti-hla-dr antibody

ABSTRACT

This invention provides an anti-HLA-DR monoclonal antibody. This invention relates to an antibody binding to HLA-DR or a functional fragment thereof having (a) life-extending effects in nonhuman animals bearing HLA-DR-expressing cancer cells and (b) activity of suppressing immune responses lower than that of L243, or an antibody binding to HLA-DR or a functional fragment thereof exhibiting immunosuppressive activity equivalent to or higher than that of the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55).

TECHNICAL FIELD

[0001] The present invention relates to an anti-HLA-DR antibody thatrecognizes the human leucocyte antigen-DR (HLA-DR), which is a cellmembrane molecule associated with immunity. Further, the presentinvention relates to the following two types of agents comprising, as anactive ingredient, the anti-HLA-DR antibody: (1) a preventive ortherapeutic agent for diseases caused by an HLA-DR-expressing cell,especially, a therapeutic agent for malignant tumors; and (2) apreventive or therapeutic agent for immune responses caused by anHLA-DR-expressing cell, especially, a therapeutic agent for chronicrheumatism.

BACKGROUND ART

[0002] The use of an antibody, which binds to a protein expressed on acell surface and is capable of leading the cell to death or toxicity,has been attempted in the treatment of cancer, etc. At present, achimeric antibody (Rituximab) targeting CD20, which is a receptorexisting on a cell membrane, and a monoclonal antibody such as ahumanized antibody targeting Her2/neu are used in the treatment ofmalignant tumors, and their therapeutic effects are acknowledged. Anantibody is characterized by a long serum half-life and high specificityfor an antigen, and thus is particularly useful as an anti-tumor agent.For example, when an antibody targeting a tumor-specific antigen isadministered, accumulation thereof in the tumor is presumed. Thus, anattack by the immune system due to the complement-dependent cytotoxicity(CDC) or antibody-dependent cellular cytotoxicity (ADCC) on cancer cellscan be expected. Binding of a radionuclide or an agent such as acytotoxic substance to the antibody enables the effective transmissionof the bound agent to a tumor site. This also reduces the amount of theagent reaching other non-specific tissues, and thus reduced side effectscan be expected. When a tumor-specific antigen has activity for inducingcell death, an agonistic antibody is administered. In contrast, when atumor-specific antigen is associated with growth and survival of cells,a neutralizing antibody is administered. This can result in theaccumulation of tumor-specific antibodies and arrest or regression oftumor growth due to the activity of the antibody. As mentioned above,antibodies are considered suitable for application as anti-tumor agentsbecause of their features.

[0003] Recently, significant anti-tumor effects of Rituximab have beenexhibited with respect to B-cell lymphoma, and the side effects thereofare limited. Since Rituximab is a chimeric human-mouse protein, however,the antigenicity of Rituximab itself is strong, an antibody against amouse moiety is produced inside the body, and the effect could bedeteriorated. For some types of cancer, the therapeutic effect ofRituximab is low with the use of Rituximab alone, and the combined usethereof with an anticancer agent is currently being clinically examined(see McLaughlin P. et. al., J Clin Oncol. (1998), 16, 2825-2833;Coiffier B. et al., Blood (1998), 92, 1927-81). Accordingly, a novelanti-tumor antibody targeting an antigen is needed, and a monoclonalantibody against HLA-DR, which is a class II major histocompatibilitycomplex (MHC) molecule, can be expected to have clinical anti-tumoractivity as an antibody that recognizes an antigen different from thatrecognized by Rituximab.

[0004] In contrast, class II major histocompatibility complex (MHC)molecules bind to antigen peptide fragments and present these antigenpeptide fragments to helper (CD4⁺) T-cells (“Th” cells) (see Babbin B.et al., Nature (1985), 317, 359-361). A monoclonal antibody that isspecific for the class II MHC molecules is reported as a very potentselective inhibitor against the immune response of Th cells in vitro(see Baxevanis C N, et. al., Immunogenetics (1980), 11, 617-625). Sincethis monoclonal antibody was discovered, it has been considered to be anagent that can be used in the selective immunosuppressive therapy ofautoimmune diseases such as chronic rheumatism. Based on the initial invivo research, significant effects of these monoclonal antibodies on Thcellular heterogeneity and autoimmune response have been elucidated (seeRosenbaum J T. et al., J. Exp. Med. (1981), 154, 1694-1702; Waldor M K.et al., Proc. Natl. Acad. Sci. USA (1983), 80, 2713-2717; Jonker M. etal., J. Autoimmun. (1988), 1, 399-414; Stevens H P. et al., Transplant.Proc. (1990), 22, 1783-1784). Further, as a result of research usingprimates, it was discovered that graft-versus-host disease in homograftwas suppressed (Billing R. & Chatterjee S. (1983), Transplant. Proc.,15, 649-650; Jonker M. et al., Transplant Proc. (1991), 23, 264-265).

[0005] Currently, immunological rejection at the time of organtransplantation is clinically suppressed using immunosuppressive agentssuch as cyclosporin A or FK506. A disadvantage of theseimmunosuppressive agents is that potent side effects are caused by thenon-specific suppression of the immune response.

[0006] Accordingly, antibodies are considered suitable for use asimmunosuppressive agents with few side effect because of their features.

DISCLOSURE OF THE INVENTION

[0007] An immunosuppressive agent using a human antibody having highimmunosuppressive activity and low immunogenicity has not yet beendeveloped.

[0008] An object of the present invention is to produce such an antibodyand use it as an anti-tumor agent or immunosuppressive agent.

[0009] The present inventors have conducted concentrated studies inorder to produce an antibody against human HLA-DR. As a result, theyhave succeeded in obtaining a monoclonal antibody exhibiting ananti-tumor effect at very low concentration on HLA-DR-expressing cancercells and a monoclonal antibody that specifically suppresses immuneactivity through HLA-DR. Further, they have identified the sequence inthe variable region of the monoclonal antibody and determined theepitope to which the monoclonal antibody binds. This has led to thecompletion of the present invention.

[0010] More specifically, the present invention is as follows.

[0011] The present invention provides, in the first aspect thereof, amonoclonal antibody that binds to HLA-DR produced from a mouse-mousehybridoma, for example, a monoclonal antibody that is preferably a humanantibody produced from HD4, HD6, HD8, or HD10, or a functional fragmentthereof. The monoclonal antibody produced from HD4, HD6, HD8, or HD10 isof an immunoglobulin G (IgG) type. The hybridoma HD8 and the hybridomaHD10 are deposited internationally at the International Patent OrganismDepositary of the National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan)as of Oct. 11, 2001 under the accession numbers FERM BP-7773 and FERMBP-7774, respectively. The hybridoma HD4 is deposited internationally atthe International Patent Organism Depositary of the National Instituteof Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) as of Oct. 11, 2001 under theaccession number FERM BP-7771. The hybridoma HD6 is depositedinternationally at the International Patent Organism Depositary of theNational Institute of Advanced Industrial Science and Technology(Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) as of Oct.11, 2001 under the accession number FERM BP-7772.

[0012] According to an embodiment of the present invention, the antibodyaccording to the present invention comprises a variable region of theantibody produced from the aforementioned hybridoma or a functionalfragment thereof.

[0013] In another embodiment of the present invention, the antibodyaccording to the present invention includes an antibody having amodified subclass, which is an antibody produced from the hybridoma HD8having an IgG1, IgG2, IgG3, or IgG4 subclass or a functional fragmentthereof, an antibody produced from the hybridoma HD4 having an IgG1,IgG2, IgG3, or IgG4 subclass or a functional fragment thereof, anantibody produced from the hybridoma HD10 having an IgG1, IgG2, IgG3, orIgG4 subclass or a functional fragment thereof, or an antibody producedfrom the hybridoma HD6 having an IgG1, IgG2, IgG3, or IgG4 subclass or afunctional fragment thereof. According to a further embodiment of thepresent invention, the antibody according to the present invention is anantibody having a modified amino acid sequence in the constant region ofthe heavy chain or a functional fragment thereof. For example, anantibody comprises amino acid 331 in the constant region of the heavychain according to the EU numbering system (see Sequences of Proteins ofImmunological Interest, NIH Publication No. 91-3242) being substitutedwith Ser or a functional fragment thereof.

[0014] In another embodiment of the present invention, the subclass ofthe antibody or a functional fragment thereof is rearranged to IgG1,IgG2, or IgG4, amino acid 331 in the constant region of the heavy chainaccording to the EU numbering system is substituted with Ser, and thesubclass is modified as IgG1, IgG1Ser, IgG2, IgG2Ser, or IgG4. As aresult, only an antibody or a functional fragment thereof having an IgG1or IgG1Ser subclass develops ADCC, and only an antibody or a functionalfragment thereof having an IgG1 or IgG2 subclass develops CDC activity.

[0015] In another aspect of the present invention, the present inventionprovides an antibody that binds to HLA-DR or a functional fragmentthereof comprising a variable region of an antibody produced from thehybridoma HD4, HD6, HD8, or HD10. In an embodiment of the presentinvention, the antibody or a functional fragment thereof according tothe present invention comprises a variable region of an antibodyproduced from the hybridoma HD8 having an amino acid sequence in themature variable region of the amino acid sequences as shown in SEQ IDNOs: 21 and 23. In another embodiment of the present invention, theantibody or a functional fragment thereof according to the presentinvention comprises a variable region of an antibody produced from thehybridoma HD4 having the amino acid sequence in the mature variableregions in the amino acid sequences as shown in SEQ ID NOs: 17 and 19.

[0016] In a further aspect of the present invention, the presentinvention relates to an antibody or a functional fragment thereof thatcan bind to a specific epitope of HLA-DR. In embodiments of the presentinvention, the antibody according to the present invention is anantibody that binds to HLA-DR or a functional fragment thereof, whichmaximally binds to the peptide as shown in SEQ ID NO: 82. This peptideis selected from among peptides that are prepared by shifting 2 aminoacids of the amino acids in the extracellular region (the amino acidsequence being shown in SEQ ID NO: 147 and the nucleotide sequence beingshown in SEQ ID NO: 146) of the HLA-DR β chain (DRB1*15011) to prepare13-mer peptides (preparing 13-mer peptides for 199 amino acids, i.e.,amino acids 29 to 227, in the amino acid sequence as shown in SEQ ID NO:147), binding the resulting peptides to a cellulose membrane through theC-terminus, and acetylating the N-terminus. Also, the antibody accordingto the present invention is an antibody that binds to HLA-DR or afunctional fragment thereof, which potently binds to all three peptidesas shown in SEQ ID NOs: 82, 83, and 84. These peptides are selected fromamong peptides that are prepared by shifting 2 amino acids of the aminoacids in the extracellular region of the HLA-DR β chain (DRB1*15011) toprepare 13-mer peptides, binding the resulting peptides to a cellulosemembrane through the C-terminus, and acetylating the N-terminus.Further, the antibody according to the present invention is an antibodythat binds to HLA-DR or a functional fragment thereof, whichsignificantly binds to all the peptides as shown in SEQ ID NOs: 24 to 39and all the peptides as shown in SEQ ID NOs: 40 to 43. These peptidesare prepared by shifting 2 amino acids of the amino acids in theextracellular region of the HLA-DR β chain (DRB1*15011) to prepare13-mer peptides, binding the resulting peptides to a cellulose membranethrough the C-terminus, and acetylating the N-terminus.

[0017] Furthermore, the present invention provides, in another aspect,an antibody or a functional fragment thereof that can extend thesurvival of individual mice by suppressing tumor growth (for example,those derived from the Raji cell transplanted to SCID mice). The amountof the antibody or a functional fragment thereof according to thepresent invention to be administered to tumor-bearing test animals (forexample, tumor-bearing test animals such as lymphoma cell-bearing mousemodels, each with a body weight of 20 g) is 0.1 μg/body to 1 μg/body or5 μg/kg to 50 μg/kg. Examples of dose are 1 μg/body or 50 μg/kg, andpreferably 0.1 μg/body or 5 μg/kg.

[0018] In an embodiment of the present invention, the antibody accordingto the present invention binds to HLA-DR or a functional fragmentthereof, which has the following properties (a) and (b):

[0019] (a) when a 6-week-old SCID mouse is inoculated intravenously with10 μl of the anti-asialo GM1 antiserum, on the next day, inoculatedintravenously with 5×10⁶ of Burkitt's lymphoma cells Raji (ATCC CCL-86),and 5 days thereafter, inoculated with 5 to 50 μg/kg, based on bodyweight, of the antibody of the present invention, the survival ratio ofthe mouse is higher than that achieved when inoculated with the sameamount of the human anti-HSA antibody; and

[0020] (b) the immunosuppressive activity is lower than that achievedwhen using the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55)at the same concentration, wherein the immunosuppressive activity isassayed as follows: 50 μl of antibody adjusted at 8 mg/mL, andpreferably 8 mg/mL using 10% FCS-containing RPMI 1640 medium is mixedwith 50 μL of mature dendritic cell suspension derived from a firsthuman donor adjusted at 2×10⁵ cells/mL using 10% FCS-containing RPMI1640 medium in wells of a 96-well plate, the mixture is allowed to standat 4° C. for 30 minutes, the resultant is mixed with 100 μL of T-cellsuspension (purity: 99% or higher) adjusted at 1×10⁶ cells/mL using 10%FCS-containing RPMI 1640 medium derived from a second human donor havinga histocompatible antigen different from that of the first human donor,the mixture is cultured at 37° C. in the presence of 5% CO₂ for 5 days,³H thymidine is added thereto at 1.0 μCi/well, the resultant is culturedat 37° C. in the presence of 5% CO₂ for 16 to 20 hours, the ³H thymidineincorporated in the cell is recovered and then measured using ascintillator, and the incorporation of the ³H thymidine into the cell isused as an indicator to assay the immunosuppressive activity.

[0021] Further, the present invention provides, in another aspectthereof, an antibody recognizing HLA-DR or a functional fragmentthereof, which exhibits immunosuppressive activity equivalent to orhigher than that of the mouse anti-HLA-DR monoclonal antibody L243 (ATCCHB-55). In an embodiment of the present invention, the antibody or afunctional fragment thereof according to the present invention has theimmunosuppressive activity equivalent to or higher than that achievedwhen using the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55)at the same concentration, wherein the immunosuppressive activity isassayed in the following manner. First, 50 μl of antibody adjusted at 8mg/mL using 10% FCS-containing RPMI 1640 medium is mixed with 50 μL ofmature dendritic cell suspension derived from a first human donoradjusted at 2×10⁵ cells/mL using 10% FCS-containing RPMI 1640 medium inwells of a 96-well plate. The mixture is then allowed to stand at 4° C.for 30 minutes, and the resultant is mixed with 100 μL of T-cellsuspension (purity: 99% or higher) adjusted at 1×10⁶ cells/mL using 10%FCS-containing RPMI 1640 medium derived from a second human donor havinga histocompatible antigen different from that of the first human donor.The resultant is cultured at 37° C. in the presence of 5% CO₂ for 5days, ³H thymidine is added thereto at 1.0 μCi/well, the resultant isfurther cultured at 37° C. in the presence of 5% CO₂ for 16 to 20 hours,the ³H thymidine incorporated in the cell is recovered and then measuredusing a scintillator, and incorporation of the ³H thymidine into thecell is used as an indicator to assay the immunosuppressive activity.

[0022] The present invention further provides, in another aspect, anucleic acid encoding an antibody comprising a variable region of anantibody produced from a hybridoma or a functional fragment thereof,wherein said nucleic acid is possessed by a hybridoma selected from thegroup consisting of the hybridoma HD8 (accession number FERM BP-7773),the hybridoma HD10 (accession number FERM BP-7774), the hybridoma HD4(accession number FERM BP-7771), and the hybridoma HD6 (accession numberFERM BP-7772), a protein encoded by the nucleic acid, an expressionvector having the nucleic acid, and a host selected from the groupconsisting of E. coli, yeast cell, insect cell, mammalian cell, plantcell, and mammalians having the expression vector. The present inventionprovides in its embodiments: a nucleic acid encoding the antibody or afunctional fragment thereof, which comprises a variable region having anamino acid sequence of the mature variable regions of the amino acidsequences as shown in SEQ ID NOs: 17 and 19; a nucleic acid encoding theantibody or a functional fragment thereof, which comprises a variableregion having an amino acid sequence of the mature variable regions ofthe amino acid sequences as shown in SEQ ID NOs: 21 and 23; and anucleic acid encoding the antibody or a functional fragment thereof,wherein the antibody is selected from the group consisting of theantibody HD8G1Ser, the antibody HD8G2Ser, and the antibody HD4G2Ser. Theantibody HD8G1Ser is the antibody HD8 having an IgG1 subclass and aminoacid 331 according to the EU numbering system being substituted withSer, the antibody HD8G2Ser is the antibody HD8 having an IgG2 subclassand amino acid 331 according to the EU numbering system beingsubstituted with Ser, and the antibody HD4G2Ser is the antibody HD4having an IgG2 subclass and amino acid 331 according to the EU numberingsystem being substituted with Ser.

[0023] The present invention further provides, in another aspectthereof, a process for producing the anti-HLA-DR monoclonal antibody,wherein a gene encoding the anti-HLA-DR monoclonal antibody is isolatedfrom a hybridoma selected from the group consisting of the hybridoma HD8(accession number FERM BP-7773), the hybridoma HD10 (accession numberFERM BP-7774), the hybridoma HD4 (accession number FERM BP-7771), andthe hybridoma HD6 (accession number FERM BP-7772), an expression vectorhaving said gene is constructed, the expression vector is introducedinto a host to express the monoclonal antibody, and the anti-HLA-DRmonoclonal antibody is collected from the resulting host, a culturesupernatant of the host, or a secretion product of the host.

[0024] The present invention further provides, in another aspectthereof, a preventive, therapeutic, or diagnostic agent for tumors,which comprises, as an active ingredient, the aforementioned antibody ora functional fragment thereof.

[0025] Examples of tumors that can be prevented or treated include atleast one member selected from the group consisting of leukemia(including chronic lymphatic leukemia and acute lymphatic leukemia),lymphoma (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-celllymphoma, B-cell lymphoma, Burkitt's lymphoma, malignant lymphoma,diffuse lymphoma, and follicular lymphoma), myeloma (including multiplemyeloma), breast cancer, colon cancer, kidney cancer, gastric cancer,ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer,esophageal cancer, liver cancer, head and neck squamous cancer, skincancer, urinary tract cancer, prostate cancer, choriocarcinoma,pharyngeal cancer, laryngeal cancer, pleural tumor, arrhenoblastoma,endometrial hyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi'ssarcoma, angioma, cavernous angioma, hemangioblastoma, retinoblastoma,spongiocytoma, neurofibroma, oligodendroglioma, medulloblastoma,neuroblastoma, neuroglioma, rhabdomyoblastoma, glioblastoma, osteogenicsarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor.

[0026] The present invention provides, in another aspect, animmunosuppressive agent comprising, as an active ingredient, theantibody or a functional fragment thereof according to the presentinvention. The present invention further provides a preventive,therapeutic, or diagnostic agent for autoimmune diseases or allergies,which comprises, as an active ingredient, the antibody or a functionalfragment thereof according to the present invention.

[0027] In an embodiment of the present invention, a preventive ortherapeutic agent is an immunosuppressive agent at the time of organtransplantation (a preventive or therapeutic agent for immunologicalrejection at the time of pancreatic islet or kidney transplantation orGVHD), a therapeutic agent for autoimmune diseases (for example,rheumatism, arteriosclerosis, multiple sclerosis, systemicerythematodes, idiopathic thrombocythemia, or Crohn's disease), or apreventive or therapeutic agent for allergic diseases such as asthma. Inanother embodiment of the present invention, the life-extending effectsare recognized in the tumor-bearing SCID mice to which the Raji cell hadbeen transplanted 5 days after the tumor transplantation with theadministration of 5 μg/kg or lower of the antibody or a functionalfragment thereof according to the present invention.

[0028] The present invention includes an antibody or a functionalfragment comprising the amino acid sequences in the mature variableregion of the heavy chain and that of the light chain of the antibodyproduced from the hybridoma HD4 as shown in SEQ ID NO: 17 or 19 and theamino acid sequences in the mature variable region of the heavy chainand that of the light chain of the antibody produced from the hybridomaHD8 as shown in SEQ ID NO: 21 or 23.

[0029] The aforementioned antibody or a functional fragment thereofcomprises, for example, the amino acid sequence in the mature variableregion of the heavy chain and that of the light chain encoded by thenucleic acid sequence isolated from the hybridoma HD4 as shown in SEQ IDNO: 16 or 18 and the amino acid sequence in the mature variable regionof the heavy chain and that of the light chain encoded by the nucleicacid sequence isolated from the hybridoma HD8 as shown in SEQ ID NO: 20or 22.

[0030] The present invention is hereafter described in detail.

[0031] It is also reported that the anti-HLA-DR monoclonal antibody hasactivity of suppressing immune responses. Based on the initial in vivoresearch, significant effects of the anti-HLA-DR monoclonal antibody onTh cellular heterogeneity and autoimmune response were elucidated (seeRosenbaum J T. et al., J. Exp. Med. (1981), 154, 1694-1702; Waldor M K.et al., Proc. Natl. Acad. Sci. USA (1983), 80, 2713-2717; Jonker M. etal., J. Autoimmun. (1988), 1, 399-414; Stevens H P. et al., Transplant.Proc. (1990), 22, 1783-1784). Further, as a result of research usingprimates, it was discovered that graft-versus-host disease in homograftwas suppressed (Billing R. & Chatterjee S. (1983), Transplant. Proc.,15, 649-650; Jonker M. et al., Transplant Proc. (1991), 23, 264-265).These reported antibodies, however, are mouse antibodies. Recently,Protein Design Labs Inc. has developed a humanized HLA-DR antibody usingthe mouse anti-HLA-DR antibody 1D10 and converting regions other thanthe variable region into the sequence of the human antibody by theirhumanizing techniques and gene recombination (see Sheri A K. et. al.,Int. J. Cancer (2001), 93, 556-565). This is clinically examined in theU.S.

[0032] The novel human anti-HLA-DR monoclonal antibody according to thepresent invention is a complete human antibody, and the antigenicityagainst the mouse sequences, which is always problematic in the mouseantibody, has already been resolved.

[0033] Any of the immunoglobulin G (IgG), immunoglobulin A (IgA),immunoglobulin E (IgE), and immunoglobulin M (IgM) antibodies can besuitably used. In general, IgG is preferable.

[0034] The terms used in the present invention are defined in order todescribe the present invention in more detail.

[0035] 1. HLA-DR and an Antibody Thereof

[0036] The antibody according to the present invention is an antibodyagainst HLA-DR, which is a class II major histocompatibility complex(MHC), i.e., an antibody that recognizes and binds to HLA-DR and anantibody that has reactivity with HLA-DR.

[0037] The “antibody that binds to HLA-DR” in the present invention isan antibody or a portion thereof having reactivity with human HLA-DR ora portion thereof, and it includes a functional fragment thereof. A“functional fragment” refers to a portion of an antibody (a partialfragment), which has at least one function of the antibody on anantigen. Specific examples thereof include F(ab′)₂, Fab′, Fab, Fv,disulphide-linked Fv, single-chain Fv (scFv), and a polymer thereof (D.J. King, Applications and Engineering of Monoclonal Antibodies, 1998, T.J. International Ltd.). Or, a “functional fragment” is a fragment of anantibody that is capable of binding to an antigen. A functional fragmentof the antibody according to the present invention binds to HLA-DR andexhibits an anti-tumor effect or potent immunosuppressive activity.

[0038] The term “human antibody” used herein refers to an antibody,which is an expression product of a human-derived antibody gene. Thehuman antibody can be obtained by introducing a human antibody locus andadministering an antigen to a transgenic animal that is capable ofproducing a human-derived antibody as described below. An example ofsuch a transgenic animal is a mouse, and a process for producing a mousethat can produce a human antibody is described in WO 02/43478.

[0039] Examples of the antibody according to the present inventioninclude various antibodies exhibiting anti-tumor effects at lowconcentration on human HLA-DR-expressing cancer cells as described inthe examples below.

[0040] The antibody according to the present invention includes amonoclonal antibody comprising the heavy chain and/or light chain havingan amino acid sequence with deletion, substitution, or addition of oneor several amino acids in various amino acid sequences thereof. Theaforementioned partial modification of amino acid (deletion,substitution, insertion, or addition) can be introduced into the aminoacid sequence of the antibody according to the present invention bypartially modifying the nucleotide sequence encoding the amino acidsequence of interest. Such partial modification of the nucleotidesequence can be introduced by a general method of conventional sitespecific mutagenesis (Proc Natl Acad Sci USA, 1984, Vol. 81: 5662). Theantibody used herein refers to an immunoglobulin in which all regionsincluding a variable region and a constant region of the heavy chain anda variable region and a constant region of the light chain constitutingthe immunoglobulin are derived from a gene encoding the immunoglobulin.

[0041] The antibody according to the present invention includes anantibody having any immunoglobulin class and isotype.

[0042] The anti-HLA-DR antibody according to the present invention canbe produced by a process as described below. Specifically, human HLA-DR,a part thereof, a binding product thereof with a suitable carriersubstance for enhancing antigenicity of the antigen (e.g., bovine serumalbumin), or the like is administered to a non-human mammalian, such asa human antibody-producing transgenic mouse, for immunization togetherwith an immunopotentiating agent (e.g., Freund's complete or incompleteadjuvant), if necessary. Alternatively, a gene encoding the human HLA-DRα chain or β chain can be introduced, and an animal cell havingexcessively expressed HLA-DR on its surface can be administered forimmunization. A monoclonal antibody can be obtained by culturing ahybridoma obtained by fusing an antibody-producing cell obtained fromthe immunized animal and a myeloma cell incapable of producing anautoantibody, and selecting a clone that produces a monoclonal antibodyhaving specific affinity to the antigen used for the immunization.

[0043] The antibody according to the present invention includes thosehaving different subclasses that were modified by genetic engineeringknown to a person skilled in the art (see, for example, EP 314161).Specifically, an antibody having a subclass that is different from theoriginal subclass can be obtained using DNA encoding a variable regionof the antibody according to the present invention using a geneticengineering technique. For example, the subclass of the antibodyaccording to the present invention can be converted into IgG2 or IgG4 toobtain an antibody having a low degree of binding to the Fc receptor. Onthe contrary, the subclass of the antibody according to the presentinvention can be converted into IgG1 or IgG3 to obtain an antibodyhaving a high degree of binding to the Fc receptor. Further,modification of the amino acid sequence in the constant region of theantibody according to the present invention by genetic engineering orbinding with a sequence of a constant region having such a sequenceenables changing of the degree of binding to the Fc receptor (seeJaneway C A. Jr. and Travers P. (1997), Immunobiology, Third Edition,Current Biology Ltd./Garland Publishing Inc.) or that to the complement(see Mi-Hua Tao, et al., 1993, J. Exp. Med). The antibody according tothe present invention includes these antibodies having modified aminoacid sequences in the constant regions. Modification of the amino acidsequence refers to deletion, substitution, or addition of one or severalamino acids of the amino acid sequence. For example, the sequence CCCencoding proline (P) 331 in the constant region of the heavy chainaccording to the EU numbering system (see Sequences of proteins ofimmunological interest, NIH Publication No. 91-3242) is varied to TCCencoding serine (S) to substitute proline with serine, thereby changingthe degree of binding to the complement. In the case of an anticanceragent, when the antibody itself does not have activity of inducing celldeath, a preferable antibody has anti-tumor activity due to theantibody-dependent cellular cytotoxicity (ADCC) or complement-dependentcytotoxicity (CDC) through the Fc receptor. When the antibody itself hasactivity of inducing cell death, an antibody having a low degree ofbinding to the Fc receptor may be preferable. In the case of animmunosuppressive agent, an antibody having no ACDD or CDC activity ispreferable when, for example, only the binding between the T-cell andthe antigen-presenting cell is three-dimensionally suppressed. When ADCCor CDC activity could cause toxicity, an antibody having resolved thetoxicity-causing activity by varying the Fc portion or changing thesubclass may be preferable.

[0044] The antibody according to the present invention includes amodified antibody, the subclass of which has been rearranged to IgG1,IgG2, or IgG4, and a modified antibody, in which amino acid 331 in theconstant region of the heavy chain of the modified antibody, thesubclass of which has been rearranged to IgG1 or IgG2, according to theEU numbering system, has been substituted with Ser to constitute IgG1Seror IgG2Ser. Specifically, the present invention includes an antibody inwhich only IgG1 and the IgG1Ser exhibit ADCC activity and only IgG1 andthe IgG2 exhibit CDC activity as a result of the subclass modificationinto IgG1, IgG1Ser, IgG2, IgG2Ser, or IgG4.

[0045] Therapeutic effects on diseases such as cancer can be furtherenhanced by binding, for example, radionuclides such as iodine, yttrium,indium, and technetium (J. W. Goding, Monoclonal Antibodies: principlesand practice, 1993, ACADEMIC PRESS), bacterial toxins such asPseudomonas exotoxin, diphtheria toxin, and ricin, chemotherapeutantssuch as methotrexate, mitomycin, and calicheamicin (D. J. King,Applications and Engineering of Monoclonal Antibodies, 1998, T. J.International Ltd, M. L. Grossbard, Monoclonal Antibody-Based Therapy ofCancer, 1998, Marcel Dekker Inc), or a prodrug such as maytansinoid(Chari et al., Cancer Res., 1992, Vol. 52: 127, Liu et al., Proc Natl.Acad Sci USA, 1996, Vol. 93: 8681) to the antibody according to thepresent invention.

[0046] In the present invention, the following steps are included in theproduction of a monoclonal antibody: (1) purification of a biopolymerthat is used as an immunogen and/or production of a cell havingexcessively expressed antigen proteins on its surface; (2) production ofan antibody-producing cell by immunizing an animal by injecting anantigen, sampling blood to assay the antibody titer, and determining thestage of excising the spleen or the like; (3) preparation of myelomacells; (4) cell fusion between an antibody-producing cell and themyeloma cell; (5) selection of a group of hybridomas producing anantibody of interest; (6) division (cloning) into a single cell clone;(7) if necessary, culture of a hybridoma for mass-producing monoclonalantibodies or breeding animals to which the hybtridoma has beentransplanted; (8) examination of physiological activity or recognitionspecificity of the thus produced monoclonal antibody or examination ofproperties thereof as labeling reagents; and the like.

[0047] A process for producing the anti-HLA-DR monoclonal antibody ishereafter described in detail along with the above steps, although theprocess for producing the antibody is not limited thereto. For example,an antibody-producing cell other than a splenic or myeloma cell can alsobe used.

[0048] (1) Purification of Antigen

[0049] A transformant is obtained by incorporating DNA encoding theHLA-DR α chain and β chain into an expression vector for animal cellsand introducing the expression vector into an animal cell. This can beused as an antigen in that state. Since the primary structures of theHLA-DR α chain and β chain are known (Steven G E, et al., (2000), TheHLA FactsBook, Academic Press), a peptide is chemically synthesized fromthe amino acid sequence of HLA-DR by a method known to a person skilledin the art, and the resultant can be used as an antigen.

[0050] A cell in which the full-length α chain and β chain of the humanHLA-DR are introduced into the L929 cell and HLA-DR heterodimers areexcessively expressed on its surface is also effective as an immunogen.pEF-neo-HLA-DRa and pEF-neo-HLA-DRβ can be produced by separatelyincorporating DNA encoding the human HLA-DR α chain protein and DNAencoding the human HLA-DR β chain protein into the expression vector foranimal cells, pEF-neo. pEF-neo is a vector comprising aneomycin-resistant gene incorporated into modified pEF-BOS (seeMizushima S. & Nagata S., Nucleic Acids Res (1990), 18, 5332). It shouldbe noted that DNA encoding HLA-DR, a vector, a host, or the like is notlimited thereto.

[0051] Specifically, a transformant obtained by transforming the L929cell in pEF-neo-HLA-DRα and pEF-neo-HLA-DRβ is cultured, andconfirmation of HLA-DR expression using the trait of neomycin resistanceacquired by the cell to which the pEF-neo vector has been inserted andthe goat anti-HLA-DR polyclonal antibody (DAKO) are employed asindicators, thereby producing the L929 cell having excessively expressedhuman HLA-DR on its surface.

[0052] (2) Step of Preparing Antibody-Producing Cell

[0053] The antigen obtained in (1) is mixed with an adjuvant such asFreund's complete or incomplete adjuvant or potash alum, andexperimental animals are immunized with the resultant as the immunogen.A transgenic mouse capable of producing a human-derived antibody is mostsuitably used as an experimental animal, and such a mouse is describedin literature by Tomizuka et al. (Tomizuka et al., Proc Natl Acad SciUSA, 2000, Vol. 97: 722).

[0054] An immunogen can be administered at the time of mouseimmunization by any of hypodermic injection, intraperitoneal injection,intravenous injection, endodermic injection, intramuscular injection, orplantar injection. Intraperitoneal injection, plantar injection, orintravenous injection is preferable.

[0055] Immunization can be performed once or several times at suitableintervals (preferably at intervals of 2 to 4 weeks). Thereafter, theantibody titer against the antigen in the serum of the immunized animalis assayed, and the animal having a sufficiently high antibody titer isused as a source of antibody-producing cells. This can enhance theeffects of the subsequent procedure. In general, the antibody-producingcell derived from an animal 3 to 5 days after the final immunization canbe preferably used for the later cell fusion.

[0056] Examples of a method for measuring the antibody titer that isused herein include various conventional techniques such asradioimmunoassay (hereafter referred to as “RIA”), enzyme-linkedimmunosorbent assay (hereafter referred to as “ELISA”), fluorescentantibody technique, and passive haemagglutination. From the viewpointsof detection sensitivity, rapidity, accuracy, the possibility ofautomating operations, and the like, the use of RIA or ELISA ispreferable.

[0057] In the present invention, the antibody titer can be assayed inthe following manner in accordance with, for example, ELISA. At theoutset, an antibody against a human antibody is allowed to adsorb on thesurface of the solid phase such as a 96-well plate for ELISA.Subsequently, the surface of the solid phase having no antigen adsorbedthereon is covered with a protein that is unrelated to an antigen, forexample, bovine serum albumin (BSA), the surface is washed, it isbrought into contact with a gradually-diluted sample (for example, mouseserum) as a primer antibody, and the anti-HLA-DR antibody in the sampleis then bound to the antigen. Further, an antibody against anenzyme-labeled human antibody is added as a secondary antibody and boundto a human antibody, followed by washing. Thereafter, the substrate ofthe enzyme is added, and changes of the absorbance, etc. are assayedbased on the coloration due to substrate decomposition. Thus, theantibody titer is calculated.

[0058] (3) Step of Preparing Myeloma Cell

[0059] A cell incapable of producing autoantibodies derived from amammalian such as a mouse, rat, guinea pig, hamster, rabbit, or humancan be used as a myeloma cell. In general, established cell linesobtained from a mouse, for example, 8-azaguanine-resistant mouse(BALB/c-derived) myeloma cells P3X63Ag8U.1 (P3-U1) (Yelton, D. E. etal., Current Topics in Microbiology and Immunology, 81, 1-7 (1978)),P3/NSI/1-Ag4-1 (NS-1) (Kohler, G. et al. European J. Immunology, 6,511-519 (1976)), Sp2/O-Ag14 (SP-2) (Shulman, M. et al. Nature, 276,269-270 (1978)), P3X63Ag8.653 (653) (Kearney, J. F. et al. J.Immunology, 123, 1548-1550 (1979)), and P3X63Ag8 (X63) (Horibata, K. andHarris, A. W. Nature, 256, 495-497 (1975)) are preferably used. Thesecell lines are subjected to subculture in suitable medium, for example,8-azaguanine medium (medium prepared by adding 8-azaguanine to RPMI-1640medium comprising glutamine, 2-mercaptoethanol, gentamicin, and fetalcalf serum (hereafter referred to as “FCS”), Iscove's ModifiedDulbecco's Medium (hereafter referred to as “IMDM”), or Dulbecco'sModified Eagle Medium (hereafter referred to as “DMEM”). These celllines are subjected to subculture in normal medium (for example, DMEMcontaining 10% FCS) 3 to 4 days before the cell fusion to reliably have2×10⁷ or more cells on the day of the cell fusion.

[0060] (4) Cell Fusion

[0061] An antibody-producing cell is a plasma cell or a lymphocyte asits precursor cell. This may be obtained from any site of an individualand can be generally obtained from, for example, the spleen, lymph node,bone marrow, tonsilla, peripheral blood, or suitable combinationsthereof, with the spleen cell being most commonly used.

[0062] After the final immunization, a site containing anantibody-producing cell therein, for example, the spleen, is excisedfrom a mouse having a predetermined antibody titer to produce a spleencell, which is an antibody-producing cell. The means for fusing thisspleen cell with the myeloma cell obtained in step (3) that is mostcommonly performed at present is a method using polyethylene glycolhaving relatively low cytotoxicity and simple fusion operations. Thismethod comprises, for example, the following procedures.

[0063] Spleen cells and myeloma cells are thoroughly washed inserum-free medium (for example, DMEM) or phosphate-buffered saline(hereafter referred to as “PBS”) and mixed with each other to bring theratio of spleen cells to myeloma cells to approximately 5:1 to 10:1,followed by centrifugation. The supernatant is removed, the precipitatedgroup of cells is thoroughly unraveled, and 1 mL of serum-free mediumcontaining 50% (w/v) polyethylene glycol (molecular weight: 1,000 to4,000) is then added thereto dropwise while stirring. Thereafter, 10 mLof serum-free medium is slowly added thereto, followed bycentrifugation. The supernatant is discarded again, the precipitatedcells are suspended in a suitable amount of normal medium containing ahypoxanthine-aminopterin-thymidine (hereafter referred to as “HAT”)solution and human interleukin 6 (hereafter referred to as “IL-6”) (thismedium is hereafter referred to as “HAT medium”), the resultant isfractionated in each well of the culture plate (hereafter referred to asa “plate”), and cultured in the presence of 5% CO₂ at 37° C. forapproximately 2 weeks. During the culture, HAT medium is suitablysupplemented.

[0064] (5) Selection of a Group of Hybridomas

[0065] When the aforementioned myeloma cells are 8-azaguanine-resistant,i.e., when they are hypoxanthine-guanine phosphoribosyltransferase(HGPRT) deficient, non-fused myeloma cells and cells fused betweenmyeloma cells cannot survive in HAT-containing medium. While cells fusedbetween antibody-producing cells or hybridomas of antibody-producingcells and myeloma cells can survive, the survival time of cells fusedbetween antibody-producing cells is limited. Accordingly, continuationof culture in HAT-containing medium results in survival of onlyhybridomas of antibody-producing cells and myeloma cells. Inconsequence, this enables the selection of hybridomas.

[0066] HAT medium for the hybridomas grown as a colony is replaced witha medium from which aminopterin has been removed (hereafter referred toas “HT medium”). Thereafter, a part of the culture supernatant iscollected to assay the anti-HLA-DR antibody titer by, for example,ELISA. When the aforementioned fusion protein is used as an antigen forELISA, an operation of eliminating a clone is required so as not toselect a clone that produces an antibody which specifically binds to theFc region of the human IgG. The presence or absence of such a clone canbe inspected by, for example, ELISA using the Fc region of the human IgGas an antigen.

[0067] A process using a 8-azaguanine-resistant cell line wasexemplified above, although other cell lines can be also used dependingon the process used for selecting a hybridoma. In such a case, thecomposition of the medium to be used is also changed.

[0068] (6) Step of Cloning

[0069] The antibody titer is assayed in the same manner as described in(2), and the hybridomas, which were found to produce specificantibodies, are transferred to the other plate to perform cloning.Examples of cloning processes include: limiting dilution in whichculture is conducted by diluting, so that one hybridoma is contained ina well of the plate; the soft agar method in which culture is conductedin a soft agar medium and colonies are recovered; a method in whichculture is conducted by removing one cell using a micromanipulator, anda “sorter clone” process, in which one cell is separated using a cellsorter. Limiting dilution is simple and often employed.

[0070] The wells, the antibody titers of which have been recognized, arerepeatedly subjected to cloning by, for example, limiting dilution 2 to4 times, and those having stable antibody titers are selected asanti-HLA-DR monoclonal antibody-producing hybridomas.

[0071] The mouse-mouse hybridomas HD8, HD10, HD4, and HD6, the humananti-HLA-DR monoclonal antibody-producing cells according to the presentinvention, are deposited internationally at the International PatentOrganism Depositary of the National Institute of Advanced IndustrialScience and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan) as of Oct. 11, 2001. The accession number of thehybridoma HD8 is FERM BP-7773, that of the hybridoma HD10 is FERMBP-7774, that of the hybridoma HD4 is FERM BP-7771, and that of thehybridoma HD6 is FERM BP-7772.

[0072] (7) Preparation of Monoclonal Antibody by Culturing Hybridomas

[0073] The hybridomas, which have completed the cloning process, arecultured in normal medium, which is a replacement of the HT medium.Large-scale culture is carried out by rotation culture in a largeculture flask, spinner culture, or culture in a hollow-fiber system. Thesupernatant obtained through this large-scale culture is purified usinga technique known to a person skilled in the art such as gel filtration.This enables the production of the anti-HLA-DR monoclonal antibody thatcomprises, as an active ingredient, the preventive or therapeutic agentof the present invention. Also, the hybridoma is multiplied in theabdominal cavity of a mouse of the same lineage (e.g., BALB/c), a nu/numouse, rat, guinea pig, hamster, or rabbit. This can provide ascitesfluid containing a large amount of the anti-HLA-DR monoclonal antibodiescomprising, as an active ingredient, the preventive or therapeutic agentof the present invention. In order to simply carry out the purification,a commercially available monoclonal antibody purification kit (e.g.,MabTrap GII Kit, Amersham Pharmacia Biotech) or the like can be used.

[0074] The thus obtained monoclonal antibody has high antigenspecificity for the human HLA-DR.

[0075] (8) Examination of Monoclonal Antibody

[0076] The isotype and the subclass of the thus obtained monoclonalantibody can be identified in the following manner. Examples of a methodfor identification include the Ouchterlony method, ELISA, and RIA.Although the Ouchterlony method is simple, this method requires aconcentrating operation when the concentration of the monoclonalantibody is low. In the case of ELISA or RIA, however, the isotype andthe subclass of the monoclonal antibody can be identified by allowingthe culture supernatant to react with the antigen-adsorbed solid phase,and using antibodies, as secondary antibodies, that correspond tovarious immunoglobulin isotypes and subclasses.

[0077] Proteins can be quantified by the Folin-Lowry method or bycalculation based on the absorbance at 280 nm (1.4 (OD280)=immunoglobulin 1 mg/ml).

[0078] The epitope recognized by the monoclonal antibody can beidentified in the following manner. Various partial constructs ofmolecules recognized by the monoclonal antibody are first prepared.Partial constructs can be prepared by, for example, a method in whichvarious partial peptides of the molecule are prepared using aconventional technique of synthesizing oligopeptides or a method inwhich a DNA sequence encoding a partial peptide of interest isincorporated in a suitable expression plasmid using a gene recombinationtechnique to produce partial constructs inside or outside a host such asE. coli. These methods are generally used in combinations to attain theabove object. For example, several polypeptide sequences, the lengths ofwhich were successively and suitably shortened from the C-terminus orN-terminus of the antigen protein, are prepared by a gene recombinationtechnique known to a person skilled in the art, and the reactivity ofthe monoclonal antibody therewith is then examined to roughly determinethe recognition site.

[0079] Thereafter, various oligopeptides in the corresponding site,variants of the peptides, or the like are synthesized using a techniqueof synthesizing oligopeptides known to a person skilled in the art. Theaffinity of the monoclonal antibody, which is an active ingredient ofthe preventive or therapeutic agent of the present invention, with thesepeptides is then inspected or competition suppressing activity ofpeptides against the binding between the monoclonal antibody and theantigen is inspected, thereby more precisely limiting the epitope. Inorder to simply obtain various oligopeptides, a commercially availablekit (for example, SPOTs Kit (Genosys Biotechnologies, Inc), a series ofMultipin Peptide Synthesis Kits using the multipin synthesis technique(Chiron Corporation), or the like) can be used.

[0080] Alternatively, a gene encoding a human monoclonal antibody iscloned from an antibody-producing cell such as a hybridoma, the cloneproduct is incorporated into a suitable vector, and the resultant isintroduced into a host (e.g., a mammalian cell strain, E. coli, yeastcell, insect cell, or plant cell) to produce a recombinant antibodyusing a gene recombination technique (P. J. Delves, ANTIBODY PRODUCTIONESSENTIAL TECHNIQUES, 1997, WILEY, P. Shepherd and C. Dean, MonoclonalAntibodies, 2000 OXFORD UNIVERSITY PRESS, J. W. Goding, MonoclonalAntibodies: principles and practice, 1993, ACADEMIC PRESS).

[0081] The present invention includes a nucleic acid comprising a genesequence of an antibody possessed by a hybridoma which produces theantibody of the present invention. More particularly, the presentinvention includes a nucleic acid that corresponds to the maturevariable regions of the heavy chain and the light chain of the antibodyproduced from the hybridoma of the present invention as described below.The nucleic acid includes DNA and RNA. The present invention includes anucleic acid, the sequence of which is modified by means ofsubstitution, deletion, and/or addition of at least one nucleotide inthe frame portion in the variable region of the heavy or light chain(see FR1, FR2, FR3, and FR4: Sequences of proteins of immunologicalinterest, NIH Publication No. 91-3242), which hybridizes with a nucleicacid complementary to a nucleic acid before sequence modification understringent conditions, binds to HLA-DR, has (a) life-extending effects inHLA-DR-expressed cancer cell-bearing non-human animals and (b) loweractivity of suppressing immune responses compared with that of L243, andencodes an antibody exhibiting immunosuppressive activity equivalent toor higher than that of the mouse anti-HLA-DR monoclonal antibody L243(ATCC HB-55) in the nucleic acid comprising a gene sequence of theantibody possessed by a hybridoma which produces the antibody of thepresent invention. The antibody refers to an immunoglobulin in which avariable region and a constant region of the heavy chain and all regionsincluding a variable region and a constant region of the light chainconstituting the immunoglobulin are derived from a gene encoding theimmunoglobulin. Stringent conditions involve the occurrence ofhybridization only when the sequence is at least 90%, preferably atleast 95%, and more preferably at least 97% homologous with the DNAsequence encoding the antibody of the present invention. In general,these conditions involve the occurrence of hybridization at atemperature about 5° C. to about 30° C., and preferably about 10° C. toabout 25° C. lower than the melting temperature of the perfect hybrid.Stringent conditions are described in J. Sambrook et al., MolecularCloning, A Laboratory Mannual, Second Edition, Cold Spring HarborLaboratory Press (1989), and the conditions described therein can beused.

[0082] In order to prepare a gene encoding the monoclonal antibody froma hybridoma, DNAs encoding the V region of the L chain, the C region ofthe L chain, the V region of the H chain, and the C region of the Hchain of the monoclonal antibody are prepared by PCR and the like. OligoDNA constructed from the anti-HLA-DR antibody gene or the amino acidsequence can be used as a primer, and DNA prepared from a hybridoma canbe used as a template. These DNAs are incorporated into a suitablevector, and the resultant is introduced into a host to be expressed.Alternatively, these DNAs are separately incorporated into each suitablevector for co-expression.

[0083] A phage or plasmid that can autonomously multiply in a hostmicroorganism is used as a vector. Examples of plasmid DNA include aplasmid derived from E. coli, Bacillus subtilis, or yeast, and anexample of phage DNA is λ phage.

[0084] A host that is used in transformation is not particularly limitedas long as the gene of interest can be expressed therein. Examplesthereof include bacteria (e.g., E. coli or Bacillus subtilis), yeast,animal cells (e.g., COS cells or CHO cells), and insect cells.

[0085] A method for introducing a gene into a host is known, andexamples thereof include any methods such as a method using calciumions, electroporation, spheroplast, the lithium acetate method, thecalcium phosphate method, and lipofection. Examples of a method forintroducing a gene into animals as described below includemicroinjection, a method for introducing a gene into the ES cell byelectroporation or lipofection, and nucleus transplantation.

[0086] In the present invention, the anti-HLA-DR antibody can beobtained by collecting it from a culture product obtained by culturing atansformant. The “culture product” refers to any of: (a) a culturesupernatant; (b) a cultured cell, cultured bacterial cell, or fragmentedproduct thereof; or (c) a secretion product of the transformant. Whenculturing a transformant, a medium that is suitable for the host ofinterest is used, and a culture method such as stationary culture orroller bottle culture is employed.

[0087] After the culture, when the protein of interest is produced in abacterial or other cell, the bacterial or other cell is destroyed tocollect the antibody. When the antibody of interest is produced outsidethe bacterial or other cell, the culture solution remaining unchanged isused, or the bacterial or other cell is removed by centrifugation, etc.Thereafter, general biochemical techniques using various types ofchromatography for isolation and purification of proteins are performedsingly or in suitable combinations. Thus, the antibody of interest canbe isolated and purified from the culture product.

[0088] With the use of a technique for preparing transgenic animals,animal hosts comprising the genes of the antibody of interest areincorporated in endogenous genes. For example, transgenic cattle, goats,sheep, or pigs are prepared. From the milk secreted from thesetransgenic animals, a large amount of monoclonal antibodies derived fromthe antibody genes thereof can be obtained (Wright, G. et al., (1991),Bio/Technology 9, 830-834). When culturing a hybridoma in vitro, ahybridoma is multiplied, maintained, and stored in accordance withvarious conditions such as properties of the cells to be cultured,purposes of tests and research, or culture methods. A known nutrientmedium that is used for producing a monoclonal antibody in a culturesupernatant or various nutrient media derived and prepared from a knownbasal medium can be used to perform the culture.

[0089] (9) Properties of the Antibody

[0090] The antibody according to the present invention has the followingfunctional properties a) and b), and these properties can be confirmedby, for example, methods described for each item:

[0091] a) HLA-DR-expressing human cancer cells are transplanted inimmunodeficient mice such as SCID mice, the survival ratio of mice wheninoculated with the antibody of the present invention is inspected, andas a result, the number of days for which the mice survive is prolonged;and

[0092] b) activity of suppressing immune responses by allogeneic mixedlymphocyte reaction is lower than that of L243. More specifically, theproperties a) and b) are as follows:

[0093] (a) when a 6-week-old SCID mouse is inoculated intravenously with10 μl of the anti-asialo GM1 antiserum, on the next day, inoculatedintravenously with 5×10⁶ of Burkitt's lymphoma cells Raji (ATCC CCL-86),and 5 days thereafter, inoculated once intravenously with 5 to 50 μg/kg,and preferably 5 μg/kg, based on body weight, of the antibody of thepresent invention, the survival ratio of the mouse 90 days after theinoculation is higher than that 90 days after the inoculation with thesame amount of the human anti-HSA antibody; and

[0094] (b) the immunosuppressive activity is lower than that achievedwhen using the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55)at the same concentration, wherein the immunosuppressive activity isassayed as follows: 50 μl of antibody adjusted at 8 to 200 mg/mL, andpreferably 8 mg/mL using 10% FCS-containing RPMI 1640 medium is mixedwith 50 μL of mature dendritic cell suspension derived from a firsthuman donor adjusted at 2×10⁵ cells/mL using 10% FCS-containing RPMI1640 medium in wells of a 96-well plate, the mixture is allowed to standat 4° C. for 30 minutes, the resultant is mixed with 100 μL of T-cellsuspension (purity: 99% or higher) adjusted at 1×10⁶ cells/mL using 10%FCS-containing RPMI 1640 medium derived from a second human donor havinga histocompatible antigen different from that of the first human donor,the mixture is cultured at 37° C. in the presence of 5% CO₂ for 5 days,³H thymidine is added thereto at 1.0 μCi/well, the resultant is culturedat 37° C. in the presence of 5% CO₂ for 16 to 20 hours, the ³H thymidineincorporated in the cell is recovered and then measured using ascintillator, and the incorporation of the ³H thymidine into the cell isused as an indicator to assay the immunosuppressive activity.

[0095] Examples of such an antibody include an antibody produced fromthe hybridoma HD4 (accession number: FERM BP-7771), an antibody producedfrom the hybridoma HD8 (accession number: FERM BP-7773), and an antibodyproduced from the hybridoma HD10 (accession number: FERM BP-7774).

[0096] The aforementioned property a) indicates that the antibody haspotent anti-tumor activity.

[0097] The antibody according to the present invention has a functionalproperty, that is, activity of suppressing immune responses byallogeneic mixed lymphocyte reaction is equivalent to or higher thanthat of L243. More specifically, the immunosuppressive activity isequivalent to or higher than that achieved when using the mouseanti-HLA-DR monoclonal antibody L243 (ATCC HB-55) at the sameconcentration, wherein the immunosuppressive activity is assayed asfollows. First, 50 μl of antibody adjusted at 8-200 mg/ml, preferably 8mg/mL using 10% FCS-containing RPMI 1640 medium is mixed with 50 μL ofmature dendritic cell suspension derived from a first human donoradjusted at 2×10⁵ cells/mL using 10% FCS-containing RPMI 1640 medium inwells of a 96-well plate. The mixture is then allowed to stand at 4° C.for 30 minutes, and the resultant is mixed with 100 μL of T-cellsuspension (purity: 99% or higher) adjusted at 1×10⁶ cells/mL using 10%FCS-containing RPMI 1640 medium derived from a second human donor havinga histocompatible antigen different from that of the first human donor.The resultant is cultured at 37° C. in the presence of 5% CO₂ for 5days, ³H thymidine is added thereto at 1.0 μCi/well, the resultant isfurther cultured at 37° C. in the presence of 5% CO₂ for 16 to 20 hours,the ³H thymidine incorporated in the cell is recovered and then measuredusing a scintillator, and incorporation of the ³H thymidine into thecell is used as an indicator to assay the immunosuppressive activity. Anexample of such an antibody is one produced from the hybridoma HD6(accession number: FERM BP-7772).

[0098] The antibody having the aforementioned activity according to thepresent invention is useful as an ingredient for a preventive ortherapeutic agent for malignant tumors or an ingredient for animmunosuppressive agent.

[0099] Even more surprisingly, the antibody according to the presentinvention significantly suppress a lowering in the survival ratio oftumor-bearing mouse models caused by the tumor cell growth at a low doseof 0.1 μg per mouse (5 μg per kg of body weight), and exhibits lifeextending-effects in mouse models. If the survival ratio of the mice towhich the antibody of the present invention has been administered issignificantly enhanced compared with that of the control mice when thehuman anti-human serum albumin (HSA) antibody is administered as acontrol simultaneously with the antibody according to the presentinvention, the antibody of the present invention can be determined toexhibit the life extending-effects. For example, the antibody accordingto the present invention and the anti-HSA antibody are administered to 5each of tumor-bearing mouse models to which lymphoma cells have beentransplanted. If at least one mouse to which the antibody of the presentinvention has been administered is alive when all of the mice in thegroup of mice to which the anti-HSA antibody has been administered havedied, it can be said that the life extending-effects can be exhibited intumor-bearing mouse models.

[0100] The immunosuppressive effects can be evaluated based on activityof suppressing the immune response by allogeneic mixed lymphocytereaction (MLR) as described above, and MLR can be carried out in aconventional manner.

[0101] Also, the epitope of the HLA-DR recognized by the antibody of thepresent invention can be identified by a conventional method. Forexample, regarding 199 amino acids in the extracellular region of theHLA-DR β chain (DRB1*15011) (199 amino acids from amino acids 29 to 227in the amino acid sequence as shown in SEQ ID NO: 147, and thenucleotide sequence encoding the amino acid sequence as shown in SEQ IDNO: 147 is shown in SEQ ID NO: 146), peptides are prepared by shiftingan amino acid in the 13-mer peptides (for example, peptides having aminoacid sequences as shown in SEQ ID NOs: 52 to 145), and the reactivity isinspected. In such a case, the antibody according to the presentinvention maximally binds to the peptide having the amino acid sequenceas shown in SEQ ID NO: 82, or potently binds to at least one of thethree peptides as shown in SEQ ID NOs: 82, 83, and 84. The term“potently binds” refers to the binding which exhibits fluorescenceintensity at least 10 times as great as that of the background in themethod as described in Example 17 (2). The antibody of the presentinvention has reactivity with peptides 61 to 71 of the HLA-DR β chain.Further, HLA-DR is known to have approximately 350 types ofpolymorphisms (see the IMGT/HLA database of EMBL-EBI, etc.). Theantibody according to the present invention is capable of recognizingsubstantially almost all of these polymorphisms. Whether or not this istrue can be determined by preparing a group of peptides includingsubstantially almost all of the approximately 350 types of polymorphismsand assaying the reactivity with substantially almost all of thesepeptides. For example, if an antibody reacts with 12 or more types ofpeptides among 16 types of peptides as shown in SEQ ID NOs: 24 to 39,this antibody is capable of recognizing substantially almost all of thepolymorphisms of the HLA-DR. The antibody according to the presentinvention significantly binds to all the peptides as shown in SEQ IDNOs: 24 to 39 and all the peptides as shown in SEQ ID NOs: 40 to 43. Theterm “significantly binds” refers to the binding which exhibitsfluorescence intensity at least 10% as great as the background in themethod as described in Example 17 (3).

[0102] 2. Pharmaceutical Composition

[0103] A preparation that comprises a purified preparation of the humananti-HLA-DR antibody of the present invention is also within the scopeof the present invention. Such a preparation preferably comprises aphysiologically acceptable diluent or carrier in addition to theantibody, and it may be a mixture of the aforementioned antibody withanother antibody or another agent such as an antibiotic. Examples of asuitable carrier include, but are not limited to, physiological saline,phosphate-buffered saline, phosphate-buffered saline glucose solution,and a buffered saline solution. Alternatively, the antibody may belyophilized and used by being recomposed with the addition of the abovebuffered aqueous solution, when it is needed. The preventive ortherapeutic agent can be administered in various dosage forms, andexamples of dosage forms include oral administration in the form of, forexample, a tablet, capsule, granule, powder, or syrup and parenteraladministration in the form of, for example, an injection, drop, orsuppository.

[0104] The dose may vary depending on symptom, age, body weight, etc. Inthe case of oral administration, the dose is generally about 0.01 mg to1,000 mg per day per adult, and this amount of preparation can beadministered in one dose or several separate doses. In the case ofparenteral administration, about 0.01 mg to 1,000 mg can be administeredper dose through hypodermic injection, intramascular injection, orintravenous injection.

[0105] The antibody or a pharmaceutical composition according to thepresent invention can be applied to the treatment or prevention ofvarious diseases or symptoms that could be caused by anHLA-DR-expressing cell. Examples of such diseases or symptoms includevarious malignant tumors, and examples of the application thereof areuse as an immunosuppressive agent at the time of organ transplantation(a preventive or therapeutic agent for immunological rejection at thetime of pancreatic islet or kidney transplantation, or GVHD), atherapeutic agent for autoimmune diseases (for example, rheumatism,arteriosclerosis, multiple sclerosis, systemic erythematodes, idiopathicthrombocythemia, or Crohn's disease), and a therapeutic agent forallergic diseases such as asthma.

[0106] For example, the antibody according to the present invention anda pharmaceutical composition comprising this antibody having thefollowing functional properties a) and b) can be used for preventing ortreating various malignant tumors:

[0107] a) HLA-DR-expressing human cancer cells are transplanted inimmunodeficient mice such as SCID mice, the survival ratio of mice wheninoculated with the antibody of the present invention is inspected, andas a result, the number of days for which the mice survive is prolonged;and

[0108] b) activity of suppressing immune responses by allogeneic mixedlymphocyte reaction is lower than that of L243.

[0109] The antibody according to the present invention and apharmaceutical composition comprising this antibody having activity ofsuppressing immune responses by allogeneic mixed lymphocyte reactionequivalent to or higher than that of L243 can be used for preventing ortreating rheumatism or graft-versus-host disease (GvHD).

[0110] The present invention includes a process for preventing ortreating the aforementioned diseases using the antibody orpharmaceutical composition according to the present invention. Thepresent invention also includes the use of the antibody according to thepresent invention in the production of a preventive or therapeutic agentfor the aforementioned diseases.

[0111] Examples of tumors that can be prevented or treated are leukemia(including chronic lymphatic leukemia and acute lymphatic leukemia),lymphoma (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, T-celllymphoma, B-cell lymphoma, Burkitt's lymphoma, malignant lymphoma,diffuse lymphoma, and follicular lymphoma), myeloma (including multiplemyeloma), breast cancer, colon cancer, kidney cancer, gastric cancer,ovarian cancer, pancreatic cancer, cervical cancer, endometrial cancer,esophageal cancer, liver cancer, head and neck squamous cancer, skincancer, urinary tract cancer, prostate cancer, choriocarcinoma,pharyngeal cancer, laryngeal cancer, pleural tumor, arrhenoblastoma,endometrial hyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi'ssarcoma, angioma, cavernous angioma, hemangioblastoma, retinoblastoma,spongiocytoma, neurofibroma, oligodendroglioma, medulloblastoma,neuroblastoma, neuroglioma, rhabdomyoblastoma, glioblastoma, osteogenicsarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor. When theantibody of the present invention is applied, the kind of a tumor is notlimited to one, and combinations of several kinds of tumors may beinvolved.

[0112] 3. Preparation Example

[0113] The molecule according to the present invention is used as anampule for an aseptic solution or suspension dissolved in water oranother pharmaceutically acceptable solution. Also, the ampule is filledwith an aseptic powder preparation (preferably lyophilized molecules ofthe present invention), and it may be diluted with a pharmaceuticallyacceptable solution at the time of use thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0114]FIG. 1 is a diagram showing the immunosuppressive activity of thepurified human anti-HLA-DR monoclonal antibody on MLR.

[0115]FIG. 2 is a diagram showing the life-extending effects of thepurified human anti-HLA-DR monoclonal antibody on the tumor-bearingmouse model. FIG. 2A shows a case where the dose is 1 μg/head, and FIG.2B shows the effects attained when the dose is 0.1 μg/head.

[0116]FIG. 3 is a diagram showing the antibody-dependent cellularcytotoxicity (ADCC) and the complement-dependent cytotoxicity (CDC) ofthe purified human anti-HLA-DR monoclonal antibody. FIG. 3A shows theADCC activities of HD8G1Ser and HD8G1, FIG. 3B shows the CDC activitiesof HD8G2CHO, HD8G1, HD8G1Ser, and HD8G4, FIG. 3C shows the ADCCactivities of HD8G1, HD8G2, HD8G2Ser, HD8G4, HD4G1, HD4G2Ser, and HD4G4,and FIG. 3D shows the CDC activities of HD8G1, HD8G2, HD8G2Ser, HD8G4,HD4G1, HD4G2Ser, and HD4G4.

[0117]FIG. 4 is a diagram showing the life-extending effects of theantibodies according to the present invention, HD8G1Ser, HD8G2Ser, andHD8G1, on the tumor-bearing mouse model. FIG. 4A shows the resultsattained when the dose is 0.1 μg per mouse, and FIG. 4B shows theresults attained when the dose is 1.0 μg per mouse.

[0118]FIG. 5 is a diagram showing the analysis of the purified humananti-HLA-DR monoclonal antibody epitope using the synthetic peptide inthe HLA-DR β chain sequence. FIGS. 5A, 5B, and 5C show analyses on HD4,HD6, and HD8, respectively.

[0119]FIG. 6 is a diagram showing the analysis of the purified humananti-HLA-DR monoclonal antibody epitope using the synthetic peptide inthe polymorphic sequence in the HLA-DR β chain epitope sequence.

FREE TEXT OF SEQUENCE LISTING

[0120] SEQ ID Nos: 1 to 15: description of artificial sequence: primer

[0121] SEQ ID Nos: 24 to 145: description of artificial sequence:peptide

BEST MODES FOR CARRYING OUT THE INVENTION

[0122] The present invention is hereafter described in more detail withreference to the following examples, although the technical scope of thepresent invention is not limited to the embodiments described in theseexamples.

EXAMPLE 1 Preparation of Antigen

[0123] In order to obtain a cell having excessively expressed humanHLA-DR on its cell membrane, plasmid vectors for the expression offull-length amino acids in the human HLA-DR a chain and β chain wereprepared. DNA encoding the HLA-DR α chain and β chain was prepared byPCR.

[0124] a) Preparation of Expression Vectors for Full-Length Human HLA-DRα Chain and β Chain

[0125] In template PCR, plasmid vectors pEF-neo-HLA-DRα andpEF-neo-HLA-DRβ holding cDNA encoding the human HLA-DR α chain and βchain were used as the templates. pEF-neo-HLA-DRα and pEF-neo-HLA-DRβwere prepared in the manner as described below. DNA of the full-lengthhuman HLA-DR α chain and that of the HLA-DR β chain were modified by thepolymerase chain reaction (PCR) for adding the EcoRI sequence at the5′-terminus and the NotI sequence and a stop codon at the 3′-terminus.PCR was carried out for 30 PCR cycles of 94° C. for 15 seconds, 55° C.for 30 seconds, and 72° C. for 60 seconds using, as a template, the cDNAderived from human peripheral blood mononuclear cells, as primers,synthesizing 5′-CCGGAATTCCCACCATGGCCATAAGTGGAGTCCCTGTG-3′ (SEQ ID NO: 1)with 5′-AAAGCGGCCGCTCATFACAGAGGCCCCCTGCGTTCTGC-3′ (SEQ ID NO: 2) for theHLA-DRα and synthesizing 5′-CCGGAATTCCTGGTCCTGTCCTGTTCTCCAGCA-3′ (SEQ IDNO: 3) with 5′-AAAGCGGCCGCTCATCAGCTCAGGAATCCTGTTGGCTG-3′ (SEQ ID NO: 4)for the HLA-DRβ, and the LA-Taq DNA polymerase (Gibco BRL). Thesynthesized sequence was isolated as the EcoRI-NotI fragment and linkedto the pEF-neo vector cleaved with the same enzyme (a vector comprisingthe neomycin-resistant gene incorporated in modified pEF-BOS (seeMizushima S. & Nagata S., Nucleic Acids Res (1990), 18, 5332)). Theresulting plasmids were designated as pEF-neo-HLA-DRα andpEF-neo-HLA-DRβ. 765 bp cDNA was encoded in the HLA-DRα incorporated inpEF-neo-HLA-DRα, and 801 bp cDNA was encoded in the HLA-DRβ incorporatedin pEF-neo-HLA-DRβ. In all of PCRs in the following examples, thereaction temperature was regulated using the Gene Amp PCR System 9700(Perkin Elmer Japan).

[0126] c) Preparation of a Human HLA-DR-Expressing Cell

[0127] pEF-neo-HLA-DRα and pEF-neo-HLA-DRβ prepared in b) wereintroduced in the L929 cell (American Type Culture Collection (ATCC) No.CCL-1) using LipofectAMINE Plus (Gibco BRL). Gene introduction wascarried out in accordance with the method described in the instructionstherefor. The gene-introduced cells were cultured in a cell cultureflask (culture area: 75 cm²) at 37° C. in the presence of 5.0% CO₂ for24 hours, and G418 (Gibco BRL) was added thereto at 1 mg/mL, followed byculture for a week. Subsequently, flow cytometry (FCM, Becton Dickinson)was carried out using the R-phycoerythrin-labeled mouseanti-human-HLA-DR antibody (BD Pharmingen), and cells having HLA-DRexpressed on the surfaces of their cell membranes were selectivelysorted from those that had attained G418-resistance among thegene-introduced cells.

[0128] All oligonucleotides such as PCR primers were synthesized using aDNA automatic synthesizer (Model: 3948, Perkin-Elmer's AppliedBiosystems Division) in accordance with the attached instructions (seeMatteucci, M. D. and Caruthers, M. H., (1981), J. Am. Chem. Soc. 103,3185-3191). After the completion of the synthesis, each of theoligonucleotides was cleaved from the support and then deprotected. Theresulting solution was exsiccated, dissolved in distilled water, andthen cryopreserved at −20° C. before use.

EXAMPLE 2 Preparation of Human Antibody-Producing Mouse

[0129] The mouse used for immunization is genetically homozygous forboth endogenous Ig heavy chain breakdown and κ light chain breakdown,and simultaneously retains a chromosome 14 fragment (SC 20) containing ahuman Ig heavy chain locus and a human Igκ chain transgene (KCo5). Thismouse was prepared by mating a mouse having a human Ig heavy chain locus(lineage A) with a mouse having a human Igκ chain transgene (lineage B).Lineage A is homozygous for both endogenous Ig heavy chain breakdown andκ light chain breakdown, and retains a chromosome 14 fragment (SC20)that is transmittable. For example, it is described in the report byTomizuka et al. (Tomizuka et al., Proc. Natl. Acad. Sci. USA, 2000, Vol.97: 722). Lineage B is homozygous for both endogenous Ig heavy chainbreakdown and κ light chain breakdown, and retains a human Igκ chaintransgene (KCo5, a transgenic mouse). It is described for example, inthe report by Fishwild et al. (Nat. Biotechnol., 1996, Vol. 114: 845).

[0130] An individual obtained by mating a male mouse of lineage A with afemale mouse of lineage B or a female mouse of lineage A with a malemouse of lineage B in which the human Ig heavy chain and the κ lightchain can be simultaneously detected in the serum (Ishida & Lonberg,IBC's 11th Antibody Engineering, Abstract 2000) was used in thefollowing immunity experiment. The aforementioned humanantibody-producing mouse (referred to as a “KM mouse”) can be obtainedfrom Kirin Brewery Co., Ltd. by making a contract.

EXAMPLE 3 Preparation of Human Monoclonal Antibody Against Human HLA-DR

[0131] In this example, a monoclonal antibody was prepared in accordancewith a common technique as described in, for example, “Tan-kuron koutaijikken sousa nyuumon (A guide to monoclonal antibody experiments)”(Tamie ANDO et al., Kodansha Ltd. Publishers, 1991). TheHLA-DR-expressing L929 cell prepared in Example 1 was used as theimmunogen human HLA-DR. A human immunoglobulin-producing humanantibody-producing mouse prepared in Example 2 was used as an animal tobe immunized.

[0132] In order to prepare a human monoclonal antibody against humanHLA-DR, human antibody-producing mice were subjected to initialimmunization intraperitoneally with the HLA-DR-expressing L929 cells(5×10⁶ cells per mouse) prepared in Example 1. After the initialimmunization, the same cells were administered for immunization 2, 4,and 8 weeks later. Three days before obtaining the spleen and the lymphnode as described below, cells (1×10⁶ cells per mouse) were administeredto the tail veins, and recombinant human IL-6 (hereafter referred to as“IL-6,” 5 ng per mouse, prepared at the Pharmaceutical ResearchLaboratory, Kirin Brewery Co., Ltd.) was administered subcutaneously.

[0133] The spleen and/or lymph node were surgically obtained from theimmunized mouse, the recovered organs were placed in 10 mL of serum-freeDMEM medium containing 350 mg/mL sodium bicarbonate, 50 units/mL ofpenicillin, and 50 μg/mL of streptomycin (Gibco BRL, hereafter referredto as “serum-free DMEM medium”), and mashed on a mesh (cell strainer,Falcon) using a spatula. The cell suspension that had passed through themesh was centrifuged to precipitate the cells. Thereafter, these cellswere washed twice in serum-free DMEM medium and then suspended inserum-free DMEM medium to count the number of cells. In contrast,myeloma cells SP2/0 (ATCC No. CRL-1581), which were cultured in DMEMmedium (Gibco BRL) containing 10% FCS (SIGMA) (this medium is hereafterreferred to as “serum-containing DMEM medium”) at 37° C. in the presenceof 5% CO₂ in such a manner that the cell concentration did not exceed1×10⁶ cells/mL, were similarly washed in serum-free DMEM medium and thensuspended in serum-free DMEM medium to count the number of cells. Therecovered cell suspension and the mouse myeloma cell suspension weremixed with each other at a ratio of 5:1, the mixture was centrifuged,and the supernatant was completely removed. 1 mL of 50% (w/v)polyethylene glycol 1500 (Boehringer Mannheim) was slowly added as afusing agent to the pellet while stirring the pellet with the tip of apipette, 1 mL of serum-free DMEM medium previously heated at 37° C. wasslowly added two separate times, and 7 mL of serum-free DMEM medium wasfurther added. After the centrifugation, the supernatant was removed,and the resulting fusion cell was subjected to screening by limitingdilution as described below. A hybridoma was selected by culturing it inDMEM medium containing 10% FCS, IL-6 (10 ng/mL), and hypoxanthine (H),aminopterin (A), and thymidine (1) (hereafter referred to as “HAT,”SIGMA). Further, a single clone was obtained by limiting dilution usingHT (SIGMA), 10% FCS, and IL-6-containing DMEM medium. Culture wasconducted in a 96-well microtiter plate (Becton Dickinson). Theselection (screening) of a hybridoma clone that produces the anti-humanHLA-DR human monoclonal antibody and the characterization of the humanmonoclonal antibody that is produced by each hybridoma were carried outwith enzyme-linked immunosorbent assay (ELISA) and FCM as describedlater.

[0134] In the screening of human monoclonal antibody-producinghybridomas, many human monoclonal antibody-producing hybridomas wereobtained, which have human immunoglobulin γ chain (hIgγ) and humanimmunoglobulin light chain κ, and which have specific reactivity withhuman HLA-DR through Cell ELISA as described in Examples 4 and 5.

EXAMPLE 4 Selection of Human Anti-HLA-DR Monoclonal Antibody-ProducingClones Having Human Immunoglobulin Light Chain κ (IgK)

[0135] Burkitt's lymphoma cells, Daudi (ATCC No. CCL-213), were added toeach well in quantities of 1×10⁵ cells, the hybridoma supernatant wasadded thereto, and the mixture was incubated at 4° C. for 20 minutes.Subsequently, the incubation product was washed twice with 1%FCS-containing PBS, the horseradish peroxidase-labeled goat anti-humanimmunoglobulin light chain κ (Igκ) antibody (50 μg/well, DAKO) was addedthereto, and the resultant was incubated at 4° C. for 20 minutes. Theincubation product was washed twice with 1% FCS-containing PBS, and 100μL each of a TMB chromogenic substrate solution (DAKO) was added to eachwell, followed by incubation at room temperature for 20 minutes. 0.5Msulfuric acid (100 μL/well) was added to each well to terminate thereaction. Absorbance at a 450 nm wavelength (reference wavelength: 570nm) was assayed using a microplate reader (1420 ARVO multilabel counter,Wallac) to select positive antibody-producing clones.

EXAMPLE 5 Identification of Subclass of Each Monoclonal Antibody

[0136] After the addition of 1×10⁵ Daudi cells to each well, thehybridoma supernatant was added thereto, and the resultant was incubatedat 4° C. for 20 minutes. Subsequently, the incubation product was washedtwice with 1% FCS-containing PBS, and horseradish peroxidase-labeledsheep anti-human IgG1 antibody, sheep anti-human IgG2 antibody, sheepanti-human IgG3 antibody, or sheep anti-human IgG4 antibody (2000-folddiluted, 50 μL/well, The Binding Site) was added to each well, followedby incubation at room temperature for 1 hour. After being washed threetimes with 1% FCS-containing PBS, a substrate buffer (TMB, 100 μL/well,DAKO) was added to each well, and incubation was carried out at roomtemperature for 20 minutes. Subsequently, 0.5M sulfuric acid (100μL/well) was added to terminate the reaction. Absorbance at a 450 nmwavelength (reference wavelength: 570 nm) was assayed using a microplatereader (1420 ARVO multilabel counter, Wallac) to identify the subclassfor each clone. Clones which were not positive for any subclass wereexcluded from the selection since they were not IgG The results only onthe finally selected clones are shown in Table 1. TABLE 1 Properties ofselected anti-HLA-DR monoclonal antibodies Activity of suppressingimmune responses by Reactivity allogeneic mixed Reactivity with L929/Subclass lymphocyte reaction L929 HLA-DR Human IgG Poly − − − HD3 IgG1++ − + HD4 IgG1 ++ − + HD6 IgG1 +++ − + HD7 IgG3 + − + HD8 IgG2 + − +HD10 IgG2 + − +

EXAMPLE 6 Process for Obtaining Normal Human Mononuclear Cell and NormalHuman Dendritic Cell

[0137] At the outset, a normal human peripheral blood-derivedmononuclear cell was prepared in accordance with a conventional methodusing Ficoll (Ficoll-PaquePLUS, Amersham Pharmacia Biotech). The normalhuman blood contained in a blood-sampling bag (Terumo) containing asodium citrate solution as an anticoagulant was centrifuged (600 G, roomtemperature, 5 minutes) to separate a cell fraction from blood plasma.The cell fraction was diluted with PBS, superposed on Ficoll, andmononuclear cells were separated by specific gravity-basedcentrifugation (400 G, room temperature, 30 minutes). The intermediatelayer was extracted as a mononuclear cell and washed twice with PBS. Theresultant was further diluted with PBS, and centrifuged at 100 G for 10minutes to remove blood platelets remaining in the supernatant. Thus,normal human peripheral blood-derived mononuclear cells (PBMC) wereobtained.

[0138] Subsequently, the obtained PBMC was allowed to react with CD14antibody attached to magnetic beads (Miltenyi Biotec (MB)) at 4° C. for30 minutes, and positive selection for CD14-positive cells was carriedout through a MACS separating column (MB). The MACS separating columnwas used in accordance with the attached instruction. The CD14-positivecells were cultured in 10% FCS-containing RPMI medium comprising GM-CSF(final concentration: 50 ng/mL, prepared at the Pharmaceutical ResearchLaboratory, Kirin Brewery Co., Ltd.) and interleukin 4 (finalconcentration: 200 ng/mL, Genzyme Corporation) for 5 to 8 days.Thereafter, lipopolysaccharide (LPS, final concentration: 40 ng/mL,Difco) was added thereto, and the resultant was cultured overnight. Theculture product was then used as a mature dendritic cell (mature DC).

EXAMPLE 7 Activity of Suppressing Immune Responses by Allogeneic MixedLymphocyte Reaction

[0139] In an allogeneic transplantation having different majorhistocompatibility antigens (MHC), the T-cell is activated byrecognizing the nonself (histoincompatible) MHC molecular complex(alloantigen), thereby generating immunological rejection. The human MHCis referred to as human leukocyte antigen (HLA), and there are class Iantigens to which HLA-A, B, and C belong, and class II antigens to whichHLA-DP, DQ, and DR belong. Further, since each molecule has apolymorphic property, several thousand combinations of human HLA arepossible. Thus, histoincompatibility is very highly likely to occur withanother person. The allogeneic mixed lymphocyte culture is a test toinspect in vitro the growth of T-cells which react with alloantigens bysubjecting lymphocytes having different histocompatible antigens(hereafter referred to as donor A and donor B for convenience) to mixedculture.

[0140] Activity of suppressing the immune response by allogeneic mixedlymphocytes was assayed using the culture supernatant of hybridomasselected in Examples 4 and 5. A normal human peripheral bloodmononuclear cell was obtained in the manner as described in Example 6.The mononuclear cells of donor A were allowed to react in RPMI 1640medium containing mitomycin C (25 μg/mL, 37° C., 30 minutes) to suppressthe multiplication of cells. After the reaction, they were washed atleast three times in RPMI 1640 medium and suspended in RPMI 1640 mediumat 1×10⁶/mL. The mononuclear cells of donor B were fractionated in a96-well plate at 1×10⁵ cell/well, the medium was removed bycentrifugation, the culture supernatant was added at 100 μL/well, andthe resultant was allowed to stand at 4° C. for 30 minutes.

[0141] Subsequently, the mononuclear cells of donor A were fractionatedat 100 μL/well in a 96-well plate containing the mononuclear cells ofdonor B and the culture supernatant, followed by culturing at 37° C. for4 days in the presence of 5% CO₂. Thereafter, ³H thymidine (AmershamPharmacia Biotech) was added thereto at 1.0 μCi/well, and culture wasfurther conducted at 37° C. for 16 to 20 hours in the presence of 5%CO₂. The ³H thymidine incorporated in cells were collected on a glassfilter mat (Printed Filtermat, Wallac) using the Micro96 Harvester(SKATRON), dehydrated, well immersed in scintillator (Betap, Scint,Wallac), and packaged. Thereafter, activity of the β dose was assayedusing a liquid scintillation counter (1205 BETAPLATE, Wallac).

[0142] Properties of the anti-human HLA-DR antibodies selected as aresult of the assay are shown in Table 1. Among those, HD8, HD10, HD4,and HD6 are deposited internationally at the International PatentOrganism Depositary of the National Institute of Advanced IndustrialScience and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan) as of Oct. 11, 2001. The accession number of thehybridoma HD8 is FERM BP-7773, that of the hybridoma HD10 is FERMBP-7774, that of the hybridoma HD4 is FERM BP-7771, and that of thehybridoma HD6 is FERM BP-7772.

EXAMPLE 8 Preparation of Each Antibody

[0143] Thu human anti-HLA-DR monoclonal antibody was purified from theculture supernatant of hybridomas obtained in Examples 4 and 5 in thefollowing manner. The culture supernatant containing the humananti-HLA-DR monoclonal antibody was subjected to affinity purificationusing rmp Protein A (Amersham Pharmacia Biotech) and a 0.8×4.0 cm column(Biorad), PBS as an adsorption buffer, and a 0.02M glycin buffer (pH 3)as an elution buffer. The elution fraction was adjusted at around pH 7.2with the addition of 1M Tris (pH 9.0). The prepared antibody solutionwas converted into PBS using a dialysis membrane (molecular weightcutoff: 10,000, Spectrum Laboratories), filter-sterilized with amembrane filter MILLEX-GV (pore diameter: 0.22 μm, MILLIPORE), and thepurified human anti-HLA-DR monoclonal antibody was obtained. Theconcentration of the purified antibody was calculated by assaying theabsorbance at 280 nm and on the basis of 1 mg/mL=1.4 OD.

[0144] The culture supernatant containing the human anti-HLA-DRmonoclonal antibody was prepared in the following manner. At the outset,the human anti-HLA-DR monoclonal antibody-producing hybridoma wasadapted to eRDF medium (Kyokuto Seiyaku) containing 10 ng/ml of IL-6 and10% fetal calf serum (FCS, SIGMA). Subsequently, a part thereof wasadapted to eRDF medium (Kyokuto Seiyaku) containing bovine insulin (5μg/ml, Gibco BRL), human transferin (5 μg/ml, Gibco BRL), ethanolamine(0.01 mM, SIGMA), sodium selenite (2.5×10⁻⁵ mM, SIGMA), and 1% low IgGFCS (HyClone) to purify the antibody. These adapted hybridomas werecryopreserved. Culture was conducted in a flask, and the culturesupernatant was collected when the rate of surviving hybridomas reached90%. The collected supernatant was applied to 10 μm- and 0.2 μm-filters(Gelman Science) to eliminate waste such as hybridomas.

EXAMPLE 9 Activity of Suppressing Immune Responses by Allogeneic MixedLymphocytes by Purified Human Anti-HLA-DR Monoclonal Antibody

[0145] The allogeneic mixed lymphocyte culture is a test to inspect invitro the growth of T-cells which react with alloantigens by subjectinglymphocytes having different histocompatible antigens (hereafterreferred to as donor A and donor B for convenience) to mixed culture.The mature dendritic cell (DC) derived from a monocyte in vitro inducedfrom the peripheral blood of donor A and the T-cells separated from theperipheral blood of donor B are exclusively subjected to mixed culture.This enables the inspection of similar reactivity of T-cells with analloantigen when the antibody-dependent cellular cytotoxicity (ADCC) ofthe macrophage, etc. has been removed. Mixed culture using allogeneic DCand T-cells was carried out in the presence of the anti-HLA-DRmonoclonal antibody to inspect the function of the anti-HLA-DRmonoclonal antibody on the T-cell alloantigen reactivity. Specifically,the mature DC of donor A was suspended in 10% FCS-containing RPMI 1640medium (RPMI-10% FCS) at 2×10⁵ cells/mL on a round-bottom, 96-well plate(Falcon). The anti-HLA-DR monoclonal antibody, the human polyclonal IgG(hIgG) as a negative control, and the mouse anti-HLA-DR monoclonalantibody L243 (ATCC HB-55) as a positive control were diluted withRPMI-10% FCS to 200, 40, and 8 mg/mL, respectively. The T-cellsseparated from the peripheral blood of donor B (purity: 99% or higher)were suspended in RPMI-10% FCS at 1×10⁶ cells/mL. At the outset, 50 μLof the mature DC derived from donor A was mixed with the same amount ofthe anti-HLA-DR monoclonal antibody on a 96-well plate, and theresultant was allowed to stand at 4° C. for 30 minutes. Subsequently,100 μL of T-cells derived from donor B was mixed therewith, cultured at37° C. for 5 days in the presence of 5% CO₂, ³H thymidine (AmershamPharmacia Biotech) was added at 1.0 μCi/well, and culture was furtherconducted at 37° C. for 16 to 20 hours in the presence of 5% CO₂. The ³Hthymidine incorporated in the cells was collected on a glass filter mat(Printed Filtermat, Wallac) using the Micro96 Harvester (SKATRON),dehydrated, well immersed in a scintillator (Betap, Scint, Wallac), andpackaged. Thereafter, activity of the β-ray dose was assayed using aliquid scintillation counter (1205 BETAPLATE, Wallac).

[0146] The results are shown in FIG. 1. The immunosuppressive activitiesof HD4, HD6, HD8, and HD10 were more dose-dependent compared with thoseof the hIgG group, those of HD8, HD4, and HD10 were lower than those ofL243, and those of HD6 were equivalent to or higher than those of thepositive control antibody L243.

[0147] This suggests that HD8, HD4, and HD10 can be used as antibodieshaving low immunosuppressive activities, and HD6 can be used as animmunosuppressive agent.

EXAMPLE 10 Examination of Reactivity of Each Monoclonal Antibody withHLA-DR-Expressing Cells

[0148] Reactivity of each purified monoclonal antibody obtained inExample 3 with the HLA-DR-expressing L929 cells prepared in Example 1was analyzed by FCM. The L929 cells and the HLA-DR-expressing L929 cellswere suspended in PBS containing 0.1% sodium azide and 1% fetal calfserum (Staining Medium, hereinafter abbreviated to “SM”) at 2×10⁷/mL,and the suspension was fractionated in a 96-well, round-bottom plate at100 μl/well. After the centrifugation (600 G, 4° C., 2 minutes), thesupernatant was removed, the culture supernatant (50 μl) of hybridomascultured in Example 3 was added, and the mixture was stirred and allowedto stand under ice cooling for 30 minutes. Centrifugation (600 G, 4° C.,2 minutes) was then carried out to remove the supernatant. The pelletwas washed twice with 100 μl/well SM, 30 μL of 0.0125 mg/mL RPEfluorescence-labeled rabbit anti-human IgK F(ab′)₂ antibody (DAKO) wasadded thereto, and the resultant was incubated under ice cooling for 30minutes. After being washed twice with SM, the incubation product wassuspended in SM, and the fluorescence intensity of each cell was assayedby FCM.

[0149] The results are shown in Table 1 above. All the antibodiesexhibited potent binding activities to HLA-DR-expressing L929 cellsalone, but none exhibited the binding activity to the L929 cell. Thisindicates that these antibodies specifically bind to HLA-DR.

EXAMPLE 11 Effect of Purified Human Anti-HLA-DR Monoclonal Antibody onTumor-Bearing Mouse Models

[0150] The effects of the purified human anti-HLA-DR monoclonal antibodyobtained in Example 8 were examined using tumor-bearing mouse models inaccordance with a method described below.

[0151] First, 5-week-old C.B-17/ICR SCID mice (CLEA Japan, Inc.) werepurchased, anti-asialo GM1 antiserum (Wako Chemicals) was diluted, and10 μL each thereof was intravenously administered to each of the micewhen they were 6 weeks old. On the next day, Burkitt's lymphoma cellsRaji (ATCC CCL-86) were intravenously administered in amounts of 5×10⁶cells per mouse. Five days after the Raji transplantation, the purifiedhuman anti-HLA-DR monoclonal antibody was administered once in tailveins of mice in amounts of 0.1 μg or 1 μg per mouse. As an antibodynegative control, the same amount of human anti-HSA antibody was used.The survival ratio after the transplantation was observed for about 3months or longer.

[0152] The results of the experiment are shown in FIG. 2. In the groupof mice to which the purified human anti-HLA-DR monoclonal antibody wasadministered in amounts of 0.1 μg per mouse, the number of survivingmice 90 days later was 3 for HD8, and 1 for HD10 and HD4 out of a groupof 5 mice (FIG. 2B). In contrast, in the group of mice to which theantibody was administered in amounts of 1.0 μg per mouse, the number ofsurviving mice was 3 for HD8, 5 for HD10, and 2 for HD 4 out of a groupof 5 mice (FIG. 2A). In the negative control group to which the anti-HSAantibody was administered, all mice died within 60 days after the Rajitransplantation.

[0153] At the time of the antibody administration, the body weight ofeach mouse was about 20 g. Thus, 0.1 μg and 1 μg per mouse means 5 μg/kgand 50 μg/kg based on body weight, respectively. This revealed that HD8exhibits an anti-tumor effect at very low dosages. Based on this and theresults attained in Example 9 together, HD8 can be said to be anantibody having low immunosuppressive activity and potent anti-tumoractivity. This suggests that HD8 can be used as an anti-cancer agentwith few side effects.

EXAMPLE 12 Preparation of a Gene Encoding a Monoclonal Antibody andConstruction of a Recombinant Antibody-Expressing Vector

[0154] (1) cDNA Cloning of HD4 and HD8 Antibody Genes and Preparation ofExpression Vector

[0155] Hybridomas HD4 and HD8 were cultured in eRDF medium (KyokutoSeiyaku) containing 10 ng/ml of IL-6 (R & D Systems) and 10% fetalbovine serum (SIGMA) and centrifuged to collect cells. Thereafter,TRIZOL (Gibco BRL) was added thereto, and total RNA was extracted inaccordance with the instructions therefor. Cloning of the variableregion in the antibody cDNA was carried out using the SMART RACE cDNAamplification Kit (Clontech) in accordance with the attachedinstruction.

[0156] First-strand cDNA was prepared using 5 μg of the total RNA as atemplate.

[0157] 1) Synthesis of First-Strand cDNA

[0158] Total RNA 5 μg/3 μL

[0159] 5′CDS 1 μL

[0160] SMART oligo 1 μL

[0161] The reaction solution having the above composition was incubatedat 70° C. for 2 minutes,

[0162] 5× Buffer 2 μL

[0163] DTT 1 μL

[0164] DNTP mix 1 μL

[0165] Superscript II 1 μL

[0166] were then added, and the mixture was incubated at 42° C. for 1.5hours.

[0167] Further, 100 μL of Tricine Buffer was added, followed byincubation at 72° C. for 7 minutes. Thus, fist-strand cDNA was obtained.

[0168] 2) Amplification of a Heavy Chain Gene and a Light Chain Gene byPCR and Construction of a Recombinant Antibody-Expressing Vector

[0169] cDNA was amplified using Z-Taq (Takara).

[0170] cDNA 2 μL

[0171] 10×Z-Taq Buffer 5 μL

[0172] dNTP mix 4 μL

[0173] Z-Taq 1 μL

[0174] Primer 1

[0175] Primer 2

[0176] The final volume of the reaction solution having the abovecomposition was brought to 50 μL with the aid of double distilled waterand then subjected to PCR.

[0177] The heavy chain was amplified by 30 PCR cycles of 98° C. for 1second and 68° C. for 30 seconds using UMP (SMART RACE cDNAamplification Kit, Clontech) and the hh-6 primer (5′-GGT CCG GGA GAT CATGAG GGT GTC CTT-3′) (SEQ ID NO: 5). Further, 1 μL of this reactionsolution was used as a template, and 20 PCR cycles of 98° C. for 1second and 68° C. for 30 seconds were repeated using NUMP (SMART RACEcDNA amplification Kit, Clontech) and the hh-3 primer (5′-GTG CAC GCCGCT GGT CAG GGC GCC TG-3′) (SEQ ID NO: 6). Thereafter, the amplified PCRproduct was purified using a PCR purification kit (QIAGEN), andnucleotide sequencing was carried out using hh-4 (5′-GGT GCC AGG GGG AAGACC GAT GG-3′) (SEQ ID NO: 7) as a primer. Based on the sequenceinformation, the HD4 heavy chain specific primer tnHD4Sal (5′-ata tgtcga cCC AGC CCT GGG ATT TTC AGG TGT TTT C-3′) (SEQ ID NO: 8) and the HD8heavy chain specific primer tnHD8Sal (5′-ata tgt cga cTGG CTG ACC AGGGCA GTC ACC AGA G-3′) (SEQ ID NO: 9) were synthesized, and the resultantprimer was used to determine the sequence also from the oppositedirection. PCR was carried out using a specific primer and tnCHNhe(5′-gat ggg ccc ttg gtg cta gct gag gag acg g-3′) (SEQ ID NO: 10) (98°C. for 1 second, 60° C. for 30 seconds, and 72° C. for 30 seconds), theamplified cDNA fragment of the heavy chain was digested with SalI andNheI, and introduced into the N5KG1-Val Lark vector (a modified vectorof IDEC Pharmaceuticals, N5KG1 (U.S. Pat. No. 6,001,358)) that wascleaved with the same enzyme. The inserted sequence was confirmed to beidentical to the one identified by direct sequencing by identifying thesequence using the vector as a template.

[0178] The light chain was amplified by repeating 30 PCR cycles of 98°C. for 1 second and 68° C. for 30 seconds using UMP (SMART RACE cDNAamplification Kit, Clontech) and the hk-2 primer (5′-GTT GAA GCT CTT TGTGAC GGG CGA GC-3′) (SEQ ID NO: 11). Further, 1 μL of this reactionsolution was used as a template, and 20 PCR cycles of 98° C. for 1second and 68° C. for 30 seconds were repeated using NUMP (SMART RACEcDNA amplification Kit, Clontech) and hk-6 (5′-TGGC GGG AAG ATG AAG ACAGAT GGT G-3′) (SEQ ID NO: 12). Thereafter, the amplified PCR product waspurified using a PCR purification kit (QIAGEN), and nucleotidesequencing was carried out using the hk-6 primer (SEQ ID NO: 12). Basedon the sequence information, the HD4 light chain specific primertnHD4Bgl (5′-ata tag atc tGC TGC TCA GTT AGG ACC CAG AGG GAA CC-3′) (SEQID NO: 13) and the HD8 light chain specific primer tnHD8Bgl (5′-ata tagatc tGG GAG TCA GAC CCA CTC AGG ACA CAG C-3′) (SEQ ID NO: 14) weresynthesized, and the resultant primer was used to determine the sequencealso from the opposite direction. PCR was carried out using a specificprimer and tnCkBsi (5′-aag aca gat ggt gca gcc acc gta cgt ttg at-3′)(SEQ ID NO: 15) (98° C. for 1 second, 60° C. for 30 seconds, and 72° C.for 30 seconds), the amplified cDNA fragment of the light chain wasdigested with BglII and BsiWI and introduced into the N5KG1-Val Larkvector that was cleaved with the same enzyme. The inserted sequence wasconfirmed to be identical to the one identified by direct sequencing byidentifying the sequence using the vector as a template.

[0179] DNAs encoding the variable region of the heavy chain, that of thelight chain of HD4, amino acid sequences of the variable region of theheavy chain, and that of the light chain of HD4 are shown below.

[0180] <The Variable Region of the Heavy Chain of HD4> (SEQ ID NO: 16)GTCGACCCAGCCCTGGGATTTTCAGGTGTTTTCAGGTGTTTTCATTTGGTGATCAGGACTGAACAGAGAGAACTCACCATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGTGAGGTGCAACTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGTGGTGGTGATAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAGAGATCATGGTTCGGGGAGTTATTATCCCTACTGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCAGCTAGC

[0181] <The Variable Region of the Heavy Chain of HD4> (SEQ ID NO: 17)MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLEWVSGISGGGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHGSGSYYPYWFDYWGQGTLVTVSSA

[0182] <The Variable Region of the Light Chain of HD4> (SEQ ID NO: 18)AGATCTGCTGCTCAGTTAGGACCCAGAGGGAACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAACTTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCCGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACG

[0183] <The Variable Region of the Light Chain of HD4> (SEQ ID NO: 19)METPAQLLFLLLLWLPDTTGELVLTQSPGTLSLSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRT

[0184] The translation initiation site in the heavy chain DNA is the ATGcodon starting from the 79th adenine (A) from the 5′-terminus of thesequence as shown in SEQ ID NO: 16, and the boundary between thevariable region and the constant region of the antibody is locatedbetween the 504th adenine (A) and the 505th guanine (G) from the5′-terminus. In the amino acid sequence, the variable region of theheavy chain is a portion between the N-terminus and the 142nd serine (S)residue in the sequence as shown in SEQ ID NO: 17, and a portioncomprising the 143rd alanine (A) and thereafter is the constant region.The N-terminus of the purified heavy chain protein was analyzed. Thisdemonstrated that the signal sequence of the heavy chain was a portionbetween the N-terminus and the 19th cysteine (C) of the sequence asshown in SEQ ID NO: 17, and the N-terminus of the matured body was the20th glutamic acid (E) of the sequence as shown in SEQ ID NO: 17.Accordingly, the matured portion in the amino acid sequence as shown inSEQ ID NO: 17 is a portion between the 20th glutamic acid and the 142ndserine.

[0185] The translation initiation site in the light chain DNA is the ATGcodon starting from the 35th A from the 5′-terminus of the sequence asshown in SEQ ID NO: 18, and the variable region is the portion betweenthe 5′-terminus and the 418th adenine (A). In the amino acid sequence,the variable region is the portion between the N-terminus and the 128thlysine (K) of the sequence as shown in SEQ ID NO: 19. The N-terminus ofthe purified light chain protein was analyzed. This demonstrated thatthe signal sequence of the light chain was the portion between theN-terminus and the 20th glycine (G) of the sequence as shown in SEQ IDNO: 19, and the N-terminus of the matured body was the 21st glutamicacid (E) of the sequence as shown in SEQ ID NO: 19. Accordingly, thematured portion in the amino acid sequence as shown in SEQ ID NO: 20 isthe portion between the 21st glutamic acid and the 128th lysine.

[0186] DNAs encoding the variable region of the heavy chain, that of thelight chain of HD8, amino acid sequences of the variable region of theheavy chain, and that of the light chain of HD8 are shown below.

[0187] <The Variable Region of the Heavy Chain of HD8> (SEQ ID NO: 20)GTCGACTGGCTGACCAGGGCAGTCACCAGAGCTCCAGACAATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTTCAGCTGCAGCACTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTTCTTGGAACTGGATCAGGCAGTCCCCATCGAGGGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTATCTGTGAAAAGTCGAATAGTCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCGAGAGAAAATTTCTATGGTTCGGAGACTTGTCATAAGAAGTATTACTGCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTA GC

[0188] <The Variable Region of the Heavy Chain of HD8> (SEQ ID NO: 21)MSVSFLIFLPVLGLPWGVLSQVQLQHSGPGLVKPSQTLSLTCAISGDSVSSNSASWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRIVINPDTSKNQFSLQLNSVTPEDTAVYYCARENFYGSETCHKKYYCYGMDVWGQGTTVTV SSAS

[0189] <The Variable Region of the Light Chain of HD8> (SEQ ID NO: 22)AGATCTGGGAGTCAGACCCACTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAACTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACG

[0190] <The Variable Region of the Light Chain of HD8> (SEQ ID NO: 23)MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTTTCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSFPLTFGGGTKVEIKRTV

[0191] The translation initiation site in the heavy chain DNA is the ATGcodon starting from the 41st adenine (A) from the 5′-terminus of thesequence as shown in SEQ ID NO: 20, and the boundary between thevariable region and the constant region of the antibody is locatedbetween the 496th adenine (A) and the 497th guanine (G) from the5′-terminus. In the amino acid sequence, the variable region of theheavy chain is the portion between the N-terminus and the 152nd serine(S) residue in the sequence as shown in SEQ ID NO: 21, and the portioncomprising the 153rd alanine (A) and thereafter is the constant region.The N-terminus of the purified heavy chain protein was analyzed. Thisdemonstrated that the signal sequence of the heavy chain was the portionbetween the N-terminus and the 20th serine (S) of the sequence as shownin SEQ ID NO: 21, and the N-terminus of the matured body was the 21stglutamine (Q) of the sequence as shown in SEQ ID NO: 21. Accordingly,the matured portion in the amino acid sequence as shown in SEQ ID NO: 21is the portion between the 21st glutamic acid and the 152nd serine.

[0192] The translation initiation site in the light chain DNA is the ATGcodon starting from the 34th A from the 5′-terminus of the sequence asshown in SEQ ID NO: 22, and the variable region is the portion betweenthe 5′-terminus and the 420th adenine (A). In the amino acid sequence,the variable region is the portion between the N-terminus and the 129thlysine (K) of the sequence as shown in SEQ ID NO: 23. The N-terminus ofthe purified light chain protein was analyzed. This demonstrated thatthe signal sequence of the light chain is the portion between theN-terminus and the 22nd cysteine (C) of the sequence as shown in SEQ IDNO: 23, and the N-terminus of the matured body was the 23rd adenine (A)of the sequence as shown in SEQ ID NO: 23. Accordingly, the maturedportion in the amino acid sequence as shown in SEQ ID NO: 23 is theportion between the 23rd adenine and the 129th lysine. TABLE 2Nucleotide sequences of synthetic DNA No Primer Sequence (5′ to 3′)Length SEQ ID NO. 1 hh-6 GGT CCG GGA GAT CAT GAG GGT GTC CCT 27 5 2 hh-3GTG CAC GCC GCT GGT CAG GGC GCC TG 26 6 3 hh-4 GGT GCC AGG GGG AAG ACCGAT GG 23 7 4 tnHD4Sal ata tgt cga cCC AGC CCT GGG ATT TTC AGG TGT TTT C37 8 5 tnHD8Sal ata tgt cga cTGG CTG ACC AGG GCA GTC ACC AGA G 35 9 6tnCHNhe gat ggg ccc ttg gtg cta gct gag gag acg g 31 10 7 hk-2 GTT GAAGCT CTT TGT GAC GGG CGA GC 26 11 8 hk-6 T GGC GGG AAG ATG AAG ACA GATGGT G 26 12 9 tnHD4Bgl ata tag atc tGC TGC TCA GTT AGG ACC CAG AGG GAACC 38 13 10 tnHD8Bgl ata tag atc tGG GAG TCA GAC CCA CTC AGG ACA CAG C37 14 11 tnCkBsi aag aca gat ggt gca gcc acc gta cgt ttg at 32 15

EXAMPLE 13 Preparation of Subclass Recombinant Vectors

[0193] The variable regions of the HD4 and HD8 antibodies wereincorporated into the following variety of vectors. The N5KG1-Val Larkvector was used for the IgG1 type, the N5KG4 Lark vector was used forthe IgG4 type (both vectors were manufactured by IDEC Pharmaceuticals,modified vectors of N5KG1 were disclosed in U.S. Pat. No. 6,001,358,N5KG1-Val Lark was modified while sustaining IgG1 as with N5KG1, andN5KG4 Lark was rearranged to the IgG4 type), a vector in which theconstant region of the heavy chain of the N5KG1-Val Lark was rearrangedinto the IgG2 type (N5KG2) was used for the IgG2 type, and the variableregions of HD4 and HD8 were incorporated into the vector in the samemanner as in Example 12. Separately, a vector in which the sequence CCCencoding proline (P) 331 according to the EU numbering system in theconstant region of the heavy chain that was incorporated into the IgG1or IgG2 vector was varied to TCC encoding serine (S) (Mi-Hua Tao et al.,1993, J. Exp. Med.) was prepared (hereafter referred to as N5KG1Ser andN5KG2Ser in that order), and the variable regions of HD4 and HD8 wereincorporated into the vector in the same manner as in Example 12. InFIG. 3 and Table 3 below, for example, the HD4 antibody having an IgG1subclass is referred to as HD4IgG1 or HD4G1, and the antibody thatexpresses the gene in which the sequence CCC encoding proline (P) 331according to the EU numbering system in the constant region of the heavychain was varied to TCC encoding serine (S) is referred to as HD4IgG1Seror HD4G1Ser. TABLE 3 Names of recombinant vectors and producedrecombinant antibodies Recombinant antibody and its cytotoxic activityAnti- Recombinant ADCC CDC body Vector Subclass antibody activityactivity HD4 N5KG1-Val Lark IgG1 HD4G1 + + HD4 N5KG2Ser IgG2Ser HD4G2Ser− − HD4 N5KG4 Lark IgG4 HD4G4 − − HD8 N5KG1-Val Lark IgG1 HD8G1 + + HD8N5KG1Ser IgG1Ser HD8G1Ser + − HD8 N5KG2 IgG2 HD8G2 − + HD8 N5KG2SerIgG2Ser HD8G2Ser − − HD8 N5KG4 Lark IgG4 HD8G4 − −

EXAMPLE 14 Preparation of Recombinant Antibody

[0194] The recombinant antibody-expressing vectors constructed inExamples 12 and 13 were introduced into host cells to prepare arecombinant antibody-expressing cell. Examples of host cells forexpression that can be used include dhfr-deficient CHO cells (ATCCCRL-9096), CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109,1999), and HEK293T (ATCC CRL-11268).

[0195] Vectors were introduced into host cells by electroporation,lipofection, or the like. Electroporation was carried out linearizingabout 2 μg of an antibody-expressing vector with a restriction enzyme,introducing the genes into 4×10⁶CHO cells using Bio-Rad electrophoreterat 350 V and 500 μF, and sowing them on a 96-well culture plate.Lipofection was carried out using LipofectAMINE Plus (Gibco BRL) inaccordance with the instructions therefor. After introducing thevectors, an agent that corresponds to the selection marker for theexpression vector was added, and the culture was continued. After thecolonies were confirmed, the antibody-expressing cells were selected bythe method described in Example 4. Antibodies were purified from theselected cells in accordance with Example 8.

EXAMPLE 15 Examination of Cytotoxic Activity

[0196] Cytotoxic activities through an antibody were assayed. Theseactivities comprised cytotoxic activity on the target cell in thepresence of the NK cell or a cell with killer activity such as aneutrophil and an antibody, i.e., antibody-dependent cellularcytotoxicity (ADCC), and cytotoxic activity on the target cell in thepresence of a complement and an antibody, i.e., complement-dependentcytotoxicity (CDC). The hybridoma-derived HD4 and HD8 antibodiesprepared in Example 8 and recombinant antibodies derived from each ofthe CHO cells prepared in Example 14 were used. In this case, hIgG wasused as a control.

[0197] In simple terms, radioactive chromium (Cr⁵¹) was incorporatedinto the cytoplasm of the target cell, and the amount of Cr⁵¹ releasedin the culture solution due to the cell death was measured based on theγ dose.

[0198] More specifically, 10⁶ Burkitt's lymphoma cells, Raji (ATCCCCL-86), as the target cells were suspended in 15 μL of fetal calf serum(FCS), 50 μL (37 MBq/mL) of Cr⁵¹-labeled sodium chromate (Perkin Elmer,hereafter referred to as “Cr⁵¹”) was added, and culture was thenconducted at 37° C. for 1 hour. Subsequently, 10 mL of medium was added,centrifugation was carried out, and the medium was discarded. Thisprocedure was repeated three times to remove Cr⁵¹ that was notincorporated in the cell.

[0199] In the ADCC assay, 200,000 mononuclear cells, which were derivedfrom the peripheral blood of a healthy person obtained by the methoddescribed in Example 6, relative to 2,000 Cr⁵¹-labeled target cells(total volume: 200 μL) were cultured in a round-bottom, 96-well plate(Falcon) together with antibodies at each concentration level at 37° C.in the presence of 5% CO₂ for 4 hours.

[0200] In the CDC assay, human serum-derived complements (finalconcentration: 5%, SIGMA) relative to 2,000 Cr⁵¹-labeled target cells(total volume: 200 μL) were cultured in a round-bottom, 96-well platetogether with antibodies at each concentration level at 37° C. in thepresence of 5% CO₂ for 2 hours.

[0201] In both of the ADCC and CDC assays, the plate was subjected tocentrifugation after the culture in order to deposit cells. Thereafter,50 μL of supernatant was transferred to a powder scintillator-containing96-well plate (Lumaplate™-96, Packard) and dehydrated at 55° C. for 1.5hours. After confirming the dehydration, the plate was covered with adedicated-purpose cover (TopSeal™-A, 96-well Microplates, Packard), andthe γ dose was measured using a scintillation counter (TopCount,Packard).

[0202] The results are shown in FIG. 3 and Table 3. HD8IgG1Ser,HD8IgG2Ser, HD8IgG4, HD4G2Ser, and HD4G4 had no CDC activity, andHD81gG1, HD8IgG2, and HD4G1 had CDC activity. Only HD8IgG1, HD8IgG1Ser,and HD4G1 had ADCC activity.

EXAMPLE 16 Effects of Recombinant Antibody on Tumor-Bearing Mouse Model

[0203] Similar models as used in Example 11 were used to examine thepharmacological function of the HD8 recombinant antibody usingtumor-bearing mouse models.

[0204] At the outset, 5-week-old C.B-17/ICR-SCID mice (CLEA Japan, Inc.)were purchased, anti-asialo GM1 antiserum (Wako Chemicals) was diluted,and 10 μL each thereof was intravenously administered to each of themice when they were 6 weeks old. On the next day, Burkitt's lymphomacells Raji (ATCC CCL-86) were intravenously administered in amounts of5×10⁶ cells per mouse. Three days after the Raji transplantation, eachantibody was administered once in tail veins of mice in amounts of 0.1μg or 1 μg per mouse. The number of surviving mice after thetransplantation was observed.

[0205] The HD8G1Ser and HD8G2Ser prepared in Example 14 wereadministered once in amounts of 0.1 or 1 μg per mouse. As an antibodynegative control, a group of mice to which the hIgG antibody used as anegative control in Example 9 was administered at 1 μg/head wasprovided. As a positive control, a group of mice to which HD8G1 wasadministered at 1 μg/mouse was provided.

[0206] The results of the above experiment are shown in FIG. 4. All thesamples died within 16 days after the Raji transplantation in thenegative control, i.e., the hIgG-administered group. The number ofsurviving mice 20 days later is as follows. With the administration of 1μg per mouse, all 6 samples survived except for the hIgG group (FIG.4A), and with the administration of 0.1 μg per mouse, all 6 samples inthe HD8G2Ser group and 2 samples in the HD8G1Ser group survived (FIG.4B). The number of surviving mice 25 days later is as follows. With theadministration of 1 μg per mouse, 2 mice in the HD8G1Ser group, 1 mousein the HD8G1 group, and 4 mice in the HD8G2Ser group survived (FIG. 4A),and with the administration of 0.1 μg per mouse, 1 mouse only in theHD8G2Ser group survived (FIG. 4B).

[0207] These results demonstrated that the HD8 recombinant antibodies,HD8G1Ser and HD8G2Ser, also exhibited anti-tumor effects at very lowdosages as with HD8. Although HD8G2Ser had neither of ADCC or CDCactivity, it exhibited beneficial effects on animal models.

EXAMPLE 17 Epitope Analyses of HD4, HD6, and HD8

[0208] The epitope of each antibody was analyzed by Western blotting inaccordance with a conventional technique. In simple terms, a celluloseor PVDF membrane was blocked with Block Ace (Yukijirushi), etc., eachantibody was allowed to react therewith at 1 μg/mL as a primary antibodyand at 0.5 μg/mL as a secondary antibody using HRP conjugated antirabbit IgG (DAKO), the HRP-labeled anti-human antibody (e.g., DAKO) wasallowed to react therewith, and the chemiluminescence was detected usinga chemiluminescent reagent (e.g., ECL Western blotting detectionreagent, Amersham Bioscience) and a chemiluminescence detector (e.g.,LAS-1000, Fuji Film).

[0209] (1) A membrane fraction was extracted from the HLA-DR-expressinglymphoma cell SKW 6.4 (ATCC TIB-215), and the HLA-DR protein waspurified from the anti-HLA-DR antibody (K28N, the name of produced cell:mouse-mouse hybridoma K28, deposited internationally at theInternational Patent Organism Depositary of the National Institute ofAdvanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) as of Feb. 22, 1994 under theaccession number of FERM BP4577) using an affinity column. The obtainedprotein was boiled using 4-20% gradient gel (Daiichi Pure Chemicals Co.,Ltd.) under nonreducing conditions (95° C., 5 minutes), electrophoresiswas carried out at a constant current of 25 mA for 1.5 hours per gel,and the resultant was transferred to a PVDF membrane at a constantcurrent of 150 mA for 1 hour per gel. Subsequently, Western blotanalysis was carried out in accordance with a conventional technique,and as a result, all of HD4, HD6, and HD8 were found to recognize theHLA-DR β chain located at approximately 30 Kda.

[0210] (2) Subsequently, regarding 199 amino acids in the extracellularregion of the HLA-DR β chain (DRB1*15011) (199 amino acids from aminoacids 29 to 227 in the amino acid sequence as shown in SEQ ID NO: 147and the nucleotide sequence encoding the amino acid sequence as shown inSEQ ID NO: 147 is shown in SEQ ID NO: 146), 94 types of peptides intotal (SEQ ID NOs: 52 to 145) were spot synthesized from the C terminuson the cellulose membrane by shifting two amino acids in the 13-merpeptides, and the N terminus was acetylated (JERINI Germany). Thesubsequent reaction was carried out based on conventional Western blotanalysis (see, for example, Reineke, U. et al., (2001), “Epitope mappingwith synthetic peptides prepared by SPOT synthesis.” AntibodyEngineering (Springer Lab Manual) Eds.: Kontermann/Dubel, 433459). Inthe analysis, LumilmagerTM (Boehringer-Mannheim) was used to representthe color intensity in each spot by numerical values.

[0211] The results are shown in FIG. 5. HD4 (FIG. 5A) exhibited potentreactivity with amino acids 61 to 73 and low reactivity with amino acids17 to 29, 63 to 75, and 65 to 77, and maximally bound to a peptidehaving the amino acid sequence as shown in SEQ ID NO: 83. HD6 (FIG. 5B)exhibited potent reactivity with amino acids 61 to 73 and low reactivitywith amino acids 57 to 69 and 59 to 71, and maximally bound to a peptidehaving the amino acid sequence as shown in SEQ ID NO: 83. HD8 (FIG. 5C)exhibited very potent reactivity with amino acids 61 to 73, 63 to 75,and 65 to 77 and potently bound to at least one peptide having the aminoacid sequences as shown in SEQ ID Nos: 82, 83, and 84. In contrast,based on the conformation of HLA-DR (see, for example, Dessen A et al.,Immunity (1997), 7, 473-481), amino acids 61 to 73 form the a helixstructure at the site where antigen-presenting peptides are retained.

[0212] (3) The β-chain polymorphisms of the 13 amino acids (amino acids61 to 73) that exhibit maximal reactivity with all of HD4, HD6, and HD8were taken into consideration, and 16 types of peptides includingsubstantially almost all of the currently known about 350 types ofpolymorphisms (see the IMGT/HLA database of EMBL-EBI, etc.) wereprepared. Further, 12 peptides (SEQ ID Nos: 40 to 51) in which eachamino acid has been substituted with alanine (guanidine if it isoriginally alanine) were subjected to Western blotting under similarconditions. In the analysis, LAS2000 and ImageGauge analyzing software(Fuji Photo Film) were used to represent the color intensity in eachspot by numerical values. FIG. 6 shows 28 types of peptide sequences andtheir reactivity with HD4, HD6, and HD8. The sequence to which eachantibody positively reacted was represented by + to ++++ depending onits intensity, and represented by − in case of negative reactivity.

[0213] Positive or negative reaction was judged in accordance with thefollowing criteria.

[0214] Less than 5% of the background: −

[0215] 5% to less than 10% of the background: +/−

[0216] 10% to less than 20% of the background: +

[0217] 20% to less than 30% of the background: ++

[0218] 30% to less than 50% of the background: +++

[0219] 50% or more of the background: ++++

[0220] HD8 positively reacted with all sequences except for those asshown in SEQ ID Nos: 48 to 51 in which amino acids 65, 66, 69, and 72had been substituted with alanine. The amino acids 65, 66, 69, and 72are conserved in substantially almost all of the discovered HLA-DR βchains (see the IMGT/HLA database of EMBL-EB1, etc.) Since they covermost of the HLA-DR β chain sequences including major sequences amongthose as shown in SEQ ID Nos: 24 to 39, HD8 is very highly likely to bea Pan-HLA-DR antibody that binds to substantially almost all the HLA-DRβ chains.

[0221] Furthermore, reactivity of HD8 was examined using the HLA-DRpositive cell strain in the same manner as in Example 10. As a result,it was found to react with ARH77 (ATCC CRL-1621), Daudi (ATCC CCL-213),HS-Sultan (ATCC CRL-1484), IM-9 (ATCC CCL-159), MC/CAR (ATCC CRL-8083),Raji (ATCC CCL-86), Ramos (ATCC CRL-1596), RL (ATCC CRL-2261), SKW6.4(ATCC TIB-215), and the L-cell (ATCC CCL-1) in which DRB1*15011/DRA*0101were forcibly expressed and 5 specimens of human peripheral bloodmononuclear cells derived from a healthy Japanese person. It has not yetbeen discovered in a cell in which nonreactive HLA-DR is expressed.Further, it reacts with 15 out of 15 crab-eating monkey specimens and 1out of 1 chimpanzee specimen. In contrast, HD8 did not react with RPMI8226 (ATCC CCL-155), which is a B-cell strain in which HLA-DR is notexpressed.

INDUSTRIAL APPLICABILITY

[0222] The present invention provides a preventive or therapeutic agentfor diseases caused by HLA-DR-expressing cells, and more particularly, amolecule that is useful as a therapeutic agent for malignant tumors forpatients having substantially almost all HLA-DR polymorphisms.

[0223] Further, the present invention provides a immunosuppressive agentfor suppressing immunological activity associated with HLA-DR, and moreparticularly, a molecule that is useful as a therapeutic agent forrheumatisms.

[0224] All publications cited herein are incorporated herein byreference in their entirety. A person skilled in the art would easilyunderstand that various modifications and changes are possible withinthe technical idea and the scope of the invention as described in theattached claims. The present invention is intended to include suchmodifications and changes.

1 147 1 38 DNA Artificial Sequence Description of ArtificialSequenceprimer 1 ccggaattcc caccatggcc ataagtggag tccctgtg 38 2 38 DNAArtificial Sequence Description of Artificial Sequenceprimer 2aaagcggccg ctcattacag aggccccctg cgttctgc 38 3 33 DNA ArtificialSequence Description of Artificial Sequenceprimer 3 ccggaattcctggtcctgtc ctgttctcca gca 33 4 38 DNA Artificial Sequence Description ofArtificial Sequenceprimer 4 aaagcggccg ctcatcagct caggaatcct gttggctg 385 27 DNA Artificial Sequence Description of Artificial Sequenceprimer 5ggtccgggag atcatgaggg tgtcctt 27 6 26 DNA Artificial SequenceDescription of Artificial Sequenceprimer 6 gtgcacgccg ctggtcaggg cgcctg26 7 23 DNA Artificial Sequence Description of Artificial Sequenceprimer7 ggtgccaggg ggaagaccga tgg 23 8 37 DNA Artificial Sequence Descriptionof Artificial Sequenceprimer 8 atatgtcgac ccagccctgg gattttcagg tgttttc37 9 35 DNA Artificial Sequence Description of Artificial Sequenceprimer9 atatgtcgac tggctgacca gggcagtcac cagag 35 10 31 DNA ArtificialSequence Description of Artificial Sequenceprimer 10 gatgggcccttggtgctagc tgaggagacg g 31 11 26 DNA Artificial Sequence Description ofArtificial Sequenceprimer 11 gttgaagctc tttgtgacgg gcgagc 26 12 26 DNAArtificial Sequence Description of Artificial Sequenceprimer 12tggcgggaag atgaagacag atggtg 26 13 38 DNA Artificial SequenceDescription of Artificial Sequenceprimer 13 atatagatct gctgctcagttaggacccag agggaacc 38 14 37 DNA Artificial Sequence Description ofArtificial Sequenceprimer 14 atatagatct gggagtcaga cccactcagg acacagc 3715 32 DNA Artificial Sequence Description of Artificial Sequenceprimer15 aagacagatg gtgcagccac cgtacgtttg at 32 16 510 DNA Homo sapiens 16gtcgacccag ccctgggatt ttcaggtgtt ttcaggtgtt ttcatttggt gatcaggact 60gaacagagag aactcaccat ggagtttggg ctgagctggc tttttcttgt ggctatttta 120aaaggtgtcc agtgtgaggt gcaactgttg gagtctgggg gaggcttggt acagcctggg 180gggtccctga gactctcctg tgcagcctct ggattcacct ttagcagcta tgccatgacc 240tgggtccgcc aggctccagg gaaggggctg gagtgggtct caggtattag tggtggtggt 300gatagcacat actacgcaga ctccgtgaag ggccggttca ccatctccag agacaattcc 360aagaacacgc tgtatctgca aatgaacagc ctgagagccg aggacacggc cgtatattac 420tgtgcgagag atcatggttc ggggagttat tatccctact ggtttgacta ctggggccag 480ggaaccctgg tcaccgtctc ctcagctagc 510 17 143 PRT Homo sapiens 17 Met GluPhe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 ValGln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 ProGly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 SerSer Tyr Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 GluTrp Val Ser Gly Ile Ser Gly Gly Gly Asp Ser Thr Tyr Tyr Ala 65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105110 Tyr Tyr Cys Ala Arg Asp His Gly Ser Gly Ser Tyr Tyr Pro Tyr Trp 115120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130135 140 18 424 DNA Homo sapiens 18 agatctgctg ctcagttagg acccagagggaaccatggaa accccagcgc agcttctctt 60 cctcctgcta ctctggctcc cagataccaccggagaactt gtgttgacgc agtctccagg 120 caccctgtct ttgtctccag gggaaagagccaccctctcc tgcagggcca gtcagagtgt 180 tagcagccgc tacttagcct ggtaccagcagaaacctggc caggctccca ggctcctcat 240 ctatggtgca tccagcaggg ccactggcatcccagacagg ttcagtggca gtgggtctgg 300 gacagacttc actctcacca tcagcagactggagcctgaa gattttgcag tgtattactg 360 tcagcagtat ggtagctcac cgctcactttcggcggaggg accaaggtgg agatcaaacg 420 tacg 424 19 130 PRT Homo sapiens 19Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 1015 Asp Thr Thr Gly Glu Leu Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 2530 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 4045 Val Ser Ser Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 5560 Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 7075 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 8590 95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr100 105 110 Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu IleLys 115 120 125 Arg Thr 130 20 502 DNA Homo sapiens 20 gtcgactggctgaccagggc agtcaccaga gctccagaca atgtctgtct ccttcctcat 60 cttcctgcccgtgctgggcc tcccatgggg tgtcctgtca caggttcagc tgcagcactc 120 aggtccaggactggtgaagc cctcgcagac cctctcactc acctgtgcca tctccgggga 180 cagtgtctctagcaacagtg cttcttggaa ctggatcagg cagtccccat cgaggggcct 240 tgagtggctgggaaggacat actacaggtc caagtggtat aatgattatg cagtatctgt 300 gaaaagtcgaatagtcatca acccagacac atccaagaac cagttctccc tgcagctgaa 360 ctctgtgactcccgaggaca cggctgtgta ttactgtgcg agagaaaatt tctatggttc 420 ggagacttgtcataagaagt attactgcta cggtatggac gtctggggcc aagggaccac 480 ggtcaccgtctcctcagcta gc 502 21 154 PRT Homo sapiens 21 Met Ser Val Ser Phe Leu IlePhe Leu Pro Val Leu Gly Leu Pro Trp 1 5 10 15 Gly Val Leu Ser Gln ValGln Leu Gln His Ser Gly Pro Gly Leu Val 20 25 30 Lys Pro Ser Gln Thr LeuSer Leu Thr Cys Ala Ile Ser Gly Asp Ser 35 40 45 Val Ser Ser Asn Ser AlaSer Trp Asn Trp Ile Arg Gln Ser Pro Ser 50 55 60 Arg Gly Leu Glu Trp LeuGly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr 65 70 75 80 Asn Asp Tyr Ala ValSer Val Lys Ser Arg Ile Val Ile Asn Pro Asp 85 90 95 Thr Ser Lys Asn GlnPhe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu 100 105 110 Asp Thr Ala ValTyr Tyr Cys Ala Arg Glu Asn Phe Tyr Gly Ser Glu 115 120 125 Thr Cys HisLys Lys Tyr Tyr Cys Tyr Gly Met Asp Val Trp Gly Gln 130 135 140 Gly ThrThr Val Thr Val Ser Ser Ala Ser 145 150 22 426 DNA Homo sapiens 22agatctggga gtcagaccca ctcaggacac agcatggaca tgagggtccc cgctcagctc 60ctggggcttc tgctgctctg gctcccaggt gccagatgtg ccatccagtt gacccagtct 120ccatcctccc tgtctgcatc tgtaggagac agagtcacca tcacttgccg ggcaagtcag 180ggcattagca gtgctttagc ctggtatcag cagaaaccag ggaaagctcc taaactcctg 240atctatgatg cctccagttt ggaaagtggg gtcccatcaa ggttcagcgg cagtggatct 300gggacagatt tcactctcac catcagcagc ctgcagcctg aagattttgc aacttattac 360tgtcaacagt ttaatagttt cccgctcact ttcggcggag ggaccaaggt ggagatcaaa 420cgtacg 426 23 132 PRT Homo sapiens 23 Met Asp Met Arg Val Pro Ala GlnLeu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys AlaIle Gln Leu Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly AspArg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Ser Ala LeuAla Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile TyrAsp Ala Ser Ser Leu Glu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln ProGlu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Phe Asn Ser Phe ProLeu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val130 24 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 24 Trp Asn Ser Gln Lys Asp Phe Leu Glu Asp Arg Arg Ala 15 10 25 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 25 Trp Asn Ser Gln Lys Asp Phe Leu Glu Arg Arg Arg Ala 15 10 26 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 26 Trp Asn Ser Gln Lys Asp Phe Leu Glu Asp Glu Arg Ala 15 10 27 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 27 Trp Asn Ser Gln Lys Asp Phe Leu Glu Gln Ala Arg Ala 15 10 28 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 28 Trp Asn Ser Gln Lys Asp Ile Leu Glu Asp Glu Arg Ala 15 10 29 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 29 Trp Asn Ser Gln Lys Asp Ile Leu Glu Gln Lys Arg Gly 15 10 30 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 30 Trp Asn Ser Gln Lys Asp Ile Leu Glu Asp Arg Arg Ala 15 10 31 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 31 Trp Asn Ser Gln Lys Asp Ile Leu Glu Asp Arg Arg Gly 15 10 32 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 32 Trp Asn Ser Gln Lys Asp Ile Leu Glu Asp Lys Arg Ala 15 10 33 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 33 Trp Asn Ser Gln Lys Asp Ile Leu Glu Gln Ala Arg Ala 15 10 34 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 34 Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala 15 10 35 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 35 Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Ala Arg Ala 15 10 36 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 36 Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Lys Arg Gly 15 10 37 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 37 Trp Asn Ser Gln Lys Asp Leu Leu Glu Asp Arg Arg Ala 15 10 38 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 38 Trp Asn Ser Gln Lys Asp Leu Leu Glu Arg Arg Arg Ala 15 10 39 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 39 Trp Asn Ser Gln Lys Asp Leu Leu Glu Asp Glu Arg Ala 15 10 40 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 40 Trp Asn Ser Gln Lys Asp Ala Leu Glu Gln Arg Arg Ala 15 10 41 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 41 Trp Asn Ser Gln Lys Asp Leu Leu Glu Ala Arg Arg Ala 15 10 42 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 42 Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Ala Arg Ala 15 10 43 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 43 Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Gly 15 10 44 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 44 Ala Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala 15 10 45 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 45 Trp Ala Ser Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala 15 10 46 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 46 Trp Asn Ala Gln Lys Asp Leu Leu Glu Gln Arg Arg Ala 15 10 47 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 47 Trp Asn Ser Ala Lys Asp Leu Leu Glu Gln Arg Arg Ala 15 10 48 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 48 Trp Asn Ser Gln Ala Asp Leu Leu Glu Gln Arg Arg Ala 15 10 49 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 49 Trp Asn Ser Gln Lys Ala Leu Leu Glu Gln Arg Arg Ala 15 10 50 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 50 Trp Asn Ser Gln Lys Asp Leu Leu Ala Gln Arg Arg Ala 15 10 51 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 51 Trp Asn Ser Gln Lys Asp Leu Leu Glu Gln Arg Ala Ala 15 10 52 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 52 Gly Asp Thr Arg Pro Arg Phe Leu Trp Gln Pro Lys Arg 15 10 53 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 53 Thr Arg Pro Arg Phe Leu Trp Gln Pro Lys Arg Glu Cys 15 10 54 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 54 Pro Arg Phe Leu Trp Gln Pro Lys Arg Glu Cys His Phe 15 10 55 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 55 Phe Leu Trp Gln Pro Lys Arg Glu Cys His Phe Phe Asn 15 10 56 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 56 Trp Gln Pro Lys Arg Glu Cys His Phe Phe Asn Gly Thr 15 10 57 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 57 Pro Lys Arg Glu Cys His Phe Phe Asn Gly Thr Glu Arg 15 10 58 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 58 Arg Glu Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg 15 10 59 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 59 Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu 15 10 60 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 60 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg 15 10 61 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 61 Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 15 10 62 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 62 Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn 15 10 63 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 63 Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu 15 10 64 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 64 Arg Phe Leu Asp Arg Tyr Phe Tyr Asn Gln Glu Glu Ser 15 10 65 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 65 Leu Asp Arg Tyr Phe Tyr Asn Gln Glu Glu Ser Val Arg 15 10 66 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 66 Arg Tyr Phe Tyr Asn Gln Glu Glu Ser Val Arg Phe Asp 15 10 67 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 67 Phe Tyr Asn Gln Glu Glu Ser Val Arg Phe Asp Ser Asp 15 10 68 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 68 Asn Gln Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly 15 10 69 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 69 Glu Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu Phe 15 10 70 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 70 Ser Val Arg Phe Asp Ser Asp Val Gly Glu Phe Arg Ala 15 10 71 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 71 Arg Phe Asp Ser Asp Val Gly Glu Phe Arg Ala Val Thr 15 10 72 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 72 Asp Ser Asp Val Gly Glu Phe Arg Ala Val Thr Glu Leu 15 10 73 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 73 Asp Val Gly Glu Phe Arg Ala Val Thr Glu Leu Gly Arg 15 10 74 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 74 Gly Glu Phe Arg Ala Val Thr Glu Leu Gly Arg Pro Asp 15 10 75 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 75 Phe Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu 15 10 76 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 76 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp 15 10 77 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 77 Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 15 10 78 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 78 Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys 15 10 79 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 79 Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Ile 15 10 80 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 80 Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Ile Leu Glu 15 10 81 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 81 Glu Tyr Trp Asn Ser Gln Lys Asp Ile Leu Glu Gln Ala 15 10 82 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 82 Trp Asn Ser Gln Lys Asp Ile Leu Glu Gln Ala Arg Ala 15 10 83 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 83 Ser Gln Lys Asp Ile Leu Glu Gln Ala Arg Ala Ala Val 15 10 84 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 84 Lys Asp Ile Leu Glu Gln Ala Arg Ala Ala Val Asp Thr 15 10 85 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 85 Ile Leu Glu Gln Ala Arg Ala Ala Val Asp Thr Tyr Cys 15 10 86 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 86 Glu Gln Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg His 15 10 87 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 87 Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr 15 10 88 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 88 Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val 15 10 89 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 89 Val Asp Thr Tyr Cys Arg His Asn Tyr Gly Val Val Glu 15 10 90 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 90 Thr Tyr Cys Arg His Asn Tyr Gly Val Val Glu Ser Phe 15 10 91 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 91 Cys Arg His Asn Tyr Gly Val Val Glu Ser Phe Thr Val 15 10 92 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 92 His Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg 15 10 93 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 93 Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg Val 15 10 94 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 94 Val Val Glu Ser Phe Thr Val Gln Arg Arg Val Gln Pro 15 10 95 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 95 Glu Ser Phe Thr Val Gln Arg Arg Val Gln Pro Lys Val 15 10 96 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 96 Phe Thr Val Gln Arg Arg Val Gln Pro Lys Val Thr Val 15 10 97 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 97 Val Gln Arg Arg Val Gln Pro Lys Val Thr Val Tyr Pro 15 10 98 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 98 Arg Arg Val Gln Pro Lys Val Thr Val Tyr Pro Ser Lys 15 10 99 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 99 Val Gln Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gln 15 10 100 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 100 Pro Lys Val Thr Val Tyr Pro Ser Lys Thr Gln Pro Leu1 5 10 101 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 101 Val Thr Val Tyr Pro Ser Lys Thr Gln Pro Leu Gln His1 5 10 102 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 102 Val Tyr Pro Ser Lys Thr Gln Pro Leu Gln His His Asn1 5 10 103 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 103 Pro Ser Lys Thr Gln Pro Leu Gln His His Asn Leu Leu1 5 10 104 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 104 Lys Thr Gln Pro Leu Gln His His Asn Leu Leu Val Cys1 5 10 105 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 105 Gln Pro Leu Gln His His Asn Leu Leu Val Cys Ser Val1 5 10 106 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 106 Leu Gln His His Asn Leu Leu Val Cys Ser Val Ser Gly1 5 10 107 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 107 His His Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr1 5 10 108 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 108 Asn Leu Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly1 5 10 109 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 109 Leu Val Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile1 5 10 110 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 110 Cys Ser Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val1 5 10 111 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 111 Val Ser Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp1 5 10 112 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 112 Gly Phe Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Leu1 5 10 113 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 113 Tyr Pro Gly Ser Ile Glu Val Arg Trp Phe Leu Asn Gly1 5 10 114 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 114 Gly Ser Ile Glu Val Arg Trp Phe Leu Asn Gly Gln Glu1 5 10 115 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 115 Ile Glu Val Arg Trp Phe Leu Asn Gly Gln Glu Glu Lys1 5 10 116 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 116 Val Arg Trp Phe Leu Asn Gly Gln Glu Glu Lys Ala Gly1 5 10 117 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 117 Trp Phe Leu Asn Gly Gln Glu Glu Lys Ala Gly Met Val1 5 10 118 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 118 Leu Asn Gly Gln Glu Glu Lys Ala Gly Met Val Ser Thr1 5 10 119 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 119 Gly Gln Glu Glu Lys Ala Gly Met Val Ser Thr Gly Leu1 5 10 120 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 120 Glu Glu Lys Ala Gly Met Val Ser Thr Gly Leu Ile Gln1 5 10 121 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 121 Lys Ala Gly Met Val Ser Thr Gly Leu Ile Gln Asn Gly1 5 10 122 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 122 Gly Met Val Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp1 5 10 123 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 123 Val Ser Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe1 5 10 124 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 124 Thr Gly Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr1 5 10 125 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 125 Leu Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val1 5 10 126 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 126 Gln Asn Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu1 5 10 127 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 127 Gly Asp Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr1 5 10 128 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 128 Trp Thr Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro1 5 10 129 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 129 Phe Gln Thr Leu Val Met Leu Glu Thr Val Pro Arg Ser1 5 10 130 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 130 Thr Leu Val Met Leu Glu Thr Val Pro Arg Ser Gly Glu1 5 10 131 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 131 Val Met Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr1 5 10 132 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 132 Leu Glu Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys1 5 10 133 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 133 Thr Val Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val1 5 10 134 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 134 Pro Arg Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His1 5 10 135 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 135 Ser Gly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser1 5 10 136 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 136 Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser Val Thr1 5 10 137 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 137 Tyr Thr Cys Gln Val Glu His Pro Ser Val Thr Ser Pro1 5 10 138 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 138 Cys Gln Val Glu His Pro Ser Val Thr Ser Pro Leu Thr1 5 10 139 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 139 Val Glu His Pro Ser Val Thr Ser Pro Leu Thr Val Glu1 5 10 140 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 140 His Pro Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg1 5 10 141 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 141 Ser Val Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg1 5 10 142 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 142 Thr Ser Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu1 5 10 143 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 143 Pro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala1 5 10 144 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 144 Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala Gln Ser1 5 10 145 13 PRT Artificial Sequence Description of ArtificialSequencepeptide 145 Val Glu Trp Arg Ala Arg Ser Glu Ser Ala Gln Ser Lys1 5 10 146 801 DNA Homo sapiens 146 atggtgtgtc tgaagctccc tggaggctcctgcatgacag cgctgacagt gacactgatg 60 gtgctgagct ccccactggc tttgtctggggacacccgac cacgtttcct gtggcagcct 120 aagagggagt gtcatttctt caatgggacggagcgggtgc ggttcctgga cagatacttc 180 tataaccagg aggagtccgt gcgcttcgacagcgacgtgg gggagttccg ggcggtgacg 240 gagctggggc ggcctgacgc tgagtactggaacagccaga aggacatcct ggagcaggcg 300 cgggccgcgg tggacaccta ctgcagacacaactacgggg ttgtggagag cttcacagtg 360 cagcggcgag tccaacctaa ggtgactgtatatccttcaa agacccagcc cctgcagcac 420 cacaacctcc tggtctgctc tgtgagtggtttctatccag gcagcattga agtcaggtgg 480 ttcctgaacg gccaggaaga gaaggctgggatggtgtcca caggcctgat ccagaatgga 540 gactggacct tccagaccct ggtgatgctggaaacagttc ctcgaagtgg agaggtttac 600 acctgccaag tggagcaccc aagcgtgacaagccctctca cagtggaatg gagagcacgg 660 tctgaatctg cacagagcaa gatgctgagtggagtcgggg gctttgtgct gggcctgctc 720 ttccttgggg ccgggctgtt catctacttcaggaatcaga aaggacactc tggacttcag 780 ccaacaggat tcctgagctg a 801 147 266PRT Homo sapiens 147 Met Val Cys Leu Lys Leu Pro Gly Gly Ser Cys Met ThrAla Leu Thr 1 5 10 15 Val Thr Leu Met Val Leu Ser Ser Pro Leu Ala LeuSer Gly Asp Thr 20 25 30 Arg Pro Arg Phe Leu Trp Gln Pro Lys Arg Glu CysHis Phe Phe Asn 35 40 45 Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr PheTyr Asn Gln Glu 50 55 60 Glu Ser Val Arg Phe Asp Ser Asp Val Gly Glu PheArg Ala Val Thr 65 70 75 80 Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp AsnSer Gln Lys Asp Ile 85 90 95 Leu Glu Gln Ala Arg Ala Ala Val Asp Thr TyrCys Arg His Asn Tyr 100 105 110 Gly Val Val Glu Ser Phe Thr Val Gln ArgArg Val Gln Pro Lys Val 115 120 125 Thr Val Tyr Pro Ser Lys Thr Gln ProLeu Gln His His Asn Leu Leu 130 135 140 Val Cys Ser Val Ser Gly Phe TyrPro Gly Ser Ile Glu Val Arg Trp 145 150 155 160 Phe Leu Asn Gly Gln GluGlu Lys Ala Gly Met Val Ser Thr Gly Leu 165 170 175 Ile Gln Asn Gly AspTrp Thr Phe Gln Thr Leu Val Met Leu Glu Thr 180 185 190 Val Pro Arg SerGly Glu Val Tyr Thr Cys Gln Val Glu His Pro Ser 195 200 205 Val Thr SerPro Leu Thr Val Glu Trp Arg Ala Arg Ser Glu Ser Ala 210 215 220 Gln SerLys Met Leu Ser Gly Val Gly Gly Phe Val Leu Gly Leu Leu 225 230 235 240Phe Leu Gly Ala Gly Leu Phe Ile Tyr Phe Arg Asn Gln Lys Gly His 245 250255 Ser Gly Leu Gln Pro Thr Gly Phe Leu Ser 260 265

1. An antibody that binds to HLA-DR or a functional fragment thereof,which is produced from the hybridoma HD8 (accession number: FERMBP-7773).
 2. An antibody that binds to HLA-DR or a functional fragmentthereof, which comprises a variable region of an antibody produced fromthe hybridoma HD8 (accession number: FERM BP-7773).
 3. The antibody or afunctional fragment thereof according to claim 2, wherein the antibodysubclass is IgG.
 4. The antibody or a functional fragment thereofaccording to claim 3, wherein IgG is IgG1.
 5. The antibody or afunctional fragment thereof according to claim 4, wherein an amino acidsequence in the constant region of the heavy chain is modified.
 6. Theantibody or a functional fragment thereof according to claim 5, whereinthe modification of the amino acid sequence in the constant region ofthe heavy chain is the substitution of amino acid 331 according to theEU numbering system with Ser.
 7. The antibody or a functional fragmentthereof according to claim 3, wherein IgG is IgG2.
 8. The antibody or afunctional fragment thereof according to claim 7, wherein an amino acidsequence in the constant region of the heavy chain is modified.
 9. Theantibody or a functional fragment thereof according to claim 8, whereinthe modification of the amino acid sequence in the constant region ofthe heavy chain is the substitution of amino acid 331 according to theEU numbering system with Ser.
 10. The antibody or a functional fragmentthereof according to claim 3, wherein IgG is IgG3.
 11. The antibody or afunctional fragment thereof according to claim 10, wherein an amino acidsequence in the constant region of the heavy chain is modified.
 12. Theantibody or a functional fragment thereof according to claim 3, whereinIgG is IgG4.
 13. The antibody or a functional fragment thereof accordingto claim 12, wherein an amino acid sequence in the constant region ofthe heavy chain is modified.
 14. The hybridoma HD8 (accession numberFERM BP-7773).
 15. An antibody that binds to HLA-DR or a functionalfragment thereof, which is produced from the hybridoma HD4 (accessionnumber: FERM BP-7771).
 16. An antibody that binds to HLA-DR or afunctional fragment thereof, which comprises a variable region of anantibody produced from the hybridoma HD4 (accession number: FERMBP-7771).
 17. The antibody or a functional fragment thereof according toclaim 16, wherein the antibody subclass is IgG.
 18. The antibody or afunctional fragment thereof according to claim 17, wherein IgG is IgG1.19. The antibody or a functional fragment thereof according to claim 18,wherein an amino acid sequence in the constant region of the heavy chainis modified.
 20. The antibody or a functional fragment thereof accordingto claim 17, wherein IgG is IgG2.
 21. The antibody or a functionalfragment thereof according to claim 20, wherein an amino acid sequencein the constant region of the heavy chain is modified.
 22. The antibodyor a functional fragment thereof according to claim 21, wherein themodification of the amino acid sequence in the constant region of theheavy chain is the substitution of amino acid 331 according to the EUnumbering system with Ser.
 23. The antibody or a functional fragmentthereof according to claim 17, wherein IgG is IgG3.
 24. The antibody ora functional fragment thereof according to claim 23, wherein an aminoacid sequence in the constant region of the heavy chain is modified. 25.The antibody or a functional fragment thereof according to claim 17,wherein IgG is IgG4.
 26. The antibody or a functional fragment thereofaccording to claim 25, wherein an amino acid sequence in the constantregion of the heavy chain is modified.
 27. The hybridoma HD4 (accessionnumber FERM BP-7771).
 28. An antibody that binds to HLA-DR or afunctional fragment thereof, which is produced from the hybridoma HD10(accession number: FERM BP-7774).
 29. The hybridoma HD10 (accessionnumber FERM BP-7774).
 30. An antibody that binds to HLA-DR or afunctional fragment thereof, which is produced from the hybridoma HD6(accession number: FERM BP-7772).
 31. An antibody that binds to HLA-DRor a functional fragment thereof, which comprises a variable region ofan antibody produced from the hybridoma HD6 (accession number: FERMBP-7772).
 32. The antibody or a functional fragment thereof according toclaim 31, wherein the antibody subclass is IgG.
 33. The antibody or afunctional fragment thereof according to claim 32, wherein IgG is IgG1.34. The antibody or a functional fragment thereof according to claim 33,wherein an amino acid sequence in the constant region of the heavy chainis modified.
 35. The antibody or a functional fragment thereof accordingto claim 32, wherein IgG is IgG2.
 36. The antibody or a functionalfragment thereof according to claim 35, wherein an amino acid sequencein the constant region of the heavy chain is modified.
 37. The antibodyor a functional fragment thereof according to claim 32, wherein IgG isIgG3.
 38. The antibody or a functional fragment thereof according toclaim 37, wherein an amino acid sequence in the constant region of theheavy chain is modified.
 39. The antibody or a functional fragmentthereof according to claim 32, wherein IgG is IgG4.
 40. The antibody ora functional fragment thereof according to claim 39, wherein an aminoacid sequence in the constant region of the heavy chain is modified. 41.The hybridoma HD6 (accession number FERM BP-7772).
 42. An antibody thatbinds to HLA-DR or a functional fragment thereof, which comprises avariable region having an amino acid sequence of the mature variableregions of the amino acid sequences as shown in SEQ ID NOs: 21 and 23.43. An antibody that binds to HLA-DR or a functional fragment thereof,which comprises a variable region comprising the mature variable regionsof peptides encoded by the nucleotide sequences as shown in SEQ ID NOs:20 and
 22. 44. The antibody or a functional fragment thereof accordingto claim 42, wherein the antibody subclass is IgG.
 45. The antibody or afunctional fragment thereof according to claim 44, wherein IgG is IgG1.46. The antibody or a functional fragment thereof according to claim 45,wherein an amino acid sequence in the constant region of the heavy chainis modified.
 47. The antibody or a functional fragment thereof accordingto claim 46, wherein the modification of the amino acid sequence in theconstant region of the heavy chain is the substitution of amino acid 331according to the EU numbering system with Ser.
 48. The antibody HD8G1Seror a functional fragment thereof, which is the antibody HD8 having anIgG1 subclass and amino acid 331 according to the EU numbering systembeing substituted with Ser.
 49. The antibody or a functional fragmentthereof according to claim 44, wherein IgG is IgG2.
 50. The antibody ora functional fragment thereof according to claim 49, wherein an aminoacid sequence in the constant region of the heavy chain is modified. 51.The antibody or a functional fragment thereof according to claim 50,wherein the modification of the amino acid sequence in the constantregion of the heavy chain is the substitution of amino acid 331according to the EU numbering system with Ser.
 52. The antibody HD8G2Seror a functional fragment thereof, which is the antibody HD8 having anIgG2 subclass and amino acid 331 according to the EU numbering systembeing substituted with Ser.
 53. The antibody or a functional fragmentthereof according to claim 44, wherein IgG is IgG3.
 54. The antibody ora functional fragment thereof according to claim 53, wherein an aminoacid sequence in the constant region of the heavy chain is modified. 55.The antibody or a functional fragment thereof according to claim 44,wherein IgG is IgG4.
 56. The antibody or a functional fragment thereofaccording to claim 55, wherein an amino acid sequence in the constantregion of the heavy chain is modified.
 57. An antibody that binds toHLA-DR or a functional fragment thereof, which comprises a variableregion having an amino acid sequence of the mature variable regions ofthe amino acid sequences as shown in SEQ ID NOs: 17 and
 19. 58. Anantibody that binds to HLA-DR or a functional fragment thereof, whichcomprises a variable region comprising the mature variable regions ofpeptides encoded by the nucleotide sequences as shown in SEQ ID NOs: 16and
 18. 59. The antibody or a functional fragment thereof according toclaim 57, wherein the antibody subclass is IgG.
 60. The antibody or afunctional fragment thereof according to claim 59, wherein IgG is IgG1.61. The antibody or a functional fragment thereof according to claim 60,wherein an amino acid sequence in the constant region of the heavy chainis modified.
 62. The antibody or a functional fragment thereof accordingto claim 59, wherein IgG is IgG2.
 63. The antibody or a functionalfragment thereof according to claim 62, wherein an amino acid sequencein the constant region of the heavy chain is modified.
 64. The antibodyor a functional fragment thereof according to claim 63, wherein themodification of the amino acid sequence in the constant region of theheavy chain is the substitution of amino acid 331 according to the EUnumbering system with Ser.
 65. The antibody HD4G2Ser or a functionalfragment thereof, which is the antibody HD4 having an IgG2 subclass andamino acid 331 according to the EU numbering system being substitutedwith Ser.
 66. The antibody or a functional fragment thereof according toclaim 59, wherein IgG is IgG3.
 67. The antibody or a functional fragmentthereof according to claim 66, wherein an amino acid sequence in theconstant region of the heavy chain is modified.
 68. The antibody or afunctional fragment thereof according to claim 59, wherein IgG is IgG4.69. The antibody or a functional fragment thereof according to claim 68,wherein an amino acid sequence in the constant region of the heavy chainis modified.
 70. An antibody that binds to HLA-DR or a functionalfragment thereof, which maximally binds to the peptide as shown in SEQID NO: 82 selected from among peptides prepared by shifting 2 aminoacids of the amino acids in the extracellular region of the HLA-DR βchain (DRB1*15011) to prepare 13-mer peptides, binding the resultingpeptides to a cellulose membrane through the C-terminus, and acetylatingthe N-terminus.
 71. An antibody that binds to HLA-DR or a functionalfragment thereof, which potently binds to all three peptides as shown inSEQ ID NOs: 82, 83, and 84 selected from among peptides prepared byshifting 2 amino acids of the amino acids in the extracellular region ofthe HLA-DR β chain (DRB1*15011) to prepare 13-mer peptides, binding theresulting peptides to a cellulose membrane through the C-terminus, andacetylating the N-terminus.
 72. An antibody that binds to HLA-DR or afunctional fragment thereof, which significantly binds to all thepeptides as shown in SEQ ID NOs: 24 to 39 and all the peptides as shownin SEQ ID NOs: 40 to 43 prepared by shifting 2 amino acids of the aminoacids in the extracellular region of the HLA-DR β chain (DRB1*15011) toprepare 13-mer peptides, binding the resulting peptides to a cellulosemembrane through the C-terminus, and acetylating the N-terminus.
 73. Anantibody that binds to HLA-DR or a functional fragment thereof, whichhas the following properties (a) and (b): (a) when a 6-week-old SCIDmouse is inoculated intravenously with 10 μl of the anti-asialo GM1antiserum, on the next day, inoculated intravenously with 5×106 ofBurkitt's lymphoma cells Raji (ATCC CCL-86), and 5 days thereafter,inoculated once with 5 μg/kg, based on body weight, of the antibody ofthe present invention, the survival ratio of the mouse 90 days after theinoculation is higher than that 90 days after the inoculation with thesame amount of the human anti-HSA antibody; and (b) theimmunosuppressive activity is lower than that achieved when using themouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55) at the sameconcentration, wherein the immunosuppressive activity is assayed asfollows: 50 μl of antibody adjusted at 8 mg/mL using the 10%FCS-containing RPMI 1640 medium is mixed with 50 μL of mature dendriticcell suspension derived from a first human donor adjusted at 2×10⁵cells/mL using the 10% FCS-containing RPMI 1640 medium in wells of a96-well plate, the mixture is allowed to stand at 4° C. for 30 minutes,the resultant is mixed with 100 μL of T-cell suspension (purity: 99% orhigher) adjusted at 1×10⁶ cells/mL using the 10% FCS-containing RPMI1640 medium derived from a second human donor having a histocompatibleantigen different from that of the first human donor, the mixture iscultured at 37° C. in the presence of 5% CO₂ for 5 days, ³H thymidine isadded thereto at 1.0 μCi/well, the resultant is cultured at 37° C. inthe presence of 5% CO₂ for 16 to 20 hours, the ³H thymidine incorporatedin the cell is recovered and then measured using a scintillator, and theincorporation of the 3H thymidine into the cell is used as an indicatorto assay the immunosuppressive activity.
 74. The antibody or afunctional fragment thereof according to claim 73, wherein the antibodyis a monoclonal antibody.
 75. The antibody or a functional fragmentthereof according to claim 73, wherein the antibody is a human antibody.76. The antibody or a functional fragment thereof according to claim 73,which is produced from a mouse-mouse hybridoma.
 77. An antibody thatbinds to HLA-DR or a functional fragment thereof, which has theimmunosuppressive activity equivalent to or higher than that achievedwhen using the mouse anti-HLA-DR monoclonal antibody L243 (ATCC HB-55)at the same concentration, wherein the immunosuppressive activity isassayed as follows: 50 μl of antibody adjusted at 8 mg/mL using the 10%FCS-containing RPMI 1640 medium is mixed with 50 μL of mature dendriticcell suspension derived from a first human donor adjusted at 2×10⁵cells/mL using the 10% FCS-containing RPMI 1640 medium in wells of a96-well plate, the mixture is allowed to stand at 4° C. for 30 minutes,the resultant is mixed with 100 μL of T-cell suspension (purity: 99% orhigher) adjusted at 1×10⁶ cells/mL using the 10% FCS-containing RPMI1640 medium derived from a second human donor having a histocompatibleantigen different from that of the first human donor, the mixture iscultured at 37° C. in the presence of 5% CO₂ for 5 days, ³H thymidine isadded thereto at 1.0 μCi/well, the resultant is cultured at 37° C. inthe presence of 5% CO₂ for 16 to 20 hours, the ³H thymidine incorporatedin the cell is recovered and then measured using a scintillator, and theincorporation of the ³H thymidine into the cell is used as an indicatorto assay the immunosuppressive activity.
 78. The antibody or afunctional fragment thereof according to claim 77, wherein the antibodyis a monoclonal antibody.
 79. The antibody or a functional fragmentthereof according to claim 77, wherein the antibody is a human antibody.80. The antibody or a functional fragment thereof according to claim 77,which is produced from a mouse-mouse hybridoma.
 81. A nucleic acidencoding an antibody comprising a variable region of an antibodyproduced from a hybridoma or a functional fragment thereof, wherein saidnucleic acid is possessed by a hybridoma selected from the groupconsisting of the hybridoma HD8 (accession number FERM BP-7773), thehybridoma HD10 (accession number FERM BP-7774), the hybridoma HD4(accession number FERM BP-7771), and the hybridoma HD6 (accession numberFERM BP-7772).
 82. A nucleic acid encoding the antibody or a functionalfragment thereof according to claim 81, which comprises a variableregion having an amino acid sequence of the mature variable regions ofthe amino acid sequences as shown in SEQ ID NOs: 17 and
 19. 83. Anucleic acid encoding the antibody or a functional fragment thereofaccording to claim 81, which comprises a variable region having an aminoacid sequence of the mature variable regions of the amino acid sequencesas shown in SEQ ID NOs: 21 and
 23. 84. A nucleic acid encoding an theantibody or a functional fragment thereof, wherein the antibody isselected from the group consisting of the antibody HD8G1Ser, which isthe antibody HD8 having an IgG1 subclass and amino acid 331 according tothe EU numbering system being substituted with Ser, the antibodyHD8G2Ser, which is the antibody HD8 having an IgG2 subclass and aminoacid 331 according to the EU numbering system being substituted withSer, and the antibody HD4G2Ser, which is the antibody HD4 having an IgG2subclass and amino acid 331 according to the EU numbering system beingsubstituted with Ser.
 85. A protein encoded by the nucleic acidaccording to any one of claims 81 to
 84. 86. An expression vector havingthe nucleic acid according to any one of claims 81 to
 84. 87. A hosthaving the expression vector according to claim
 86. 88. The hostaccording to claim 87, which is selected from the group consisting of E.coli, yeast cell, insect cell, mammalian animal cell, plant cell, andmammalian animals.
 89. A process for producing the anti-HLA-DRmonoclonal antibody, wherein a gene encoding the anti-HLA-DR monoclonalantibody is isolated from a hybridoma selected from the group consistingof the hybridoma HD8 (accession number FERM BP-7773), the hybridoma HD10(accession number FERM BP-7774), the hybridoma HD4 (accession numberFERM BP-7771), and the hybridoma HD6 (accession number FERM BP-7772), anexpression vector having said gene is constructed, the expression vectoris introduced into a host to express the monoclonal antibody, and theanti-HLA-DR monoclonal antibody is collected from the resulting host, aculture supernatant of the host, or a secretion product of the host. 90.A preventive, therapeutic, or diagnostic agent for tumors, whichcomprises, as an active ingredient, the antibody or a functionalfragment thereof according to any one of claims 1 to 26, 28, and 42 to76.
 91. The preventive, therapeutic, or diagnostic agent for tumorsaccording to claim 90, wherein the tumor is at least one member selectedfrom the group consisting of leukemia (including chronic lymphaticleukemia and acute lymphatic leukemia), lymphoma (includingnon-Hodgkin's lymphoma, Hodgkin's lymphoma, T-cell lymphoma, B-celllymphoma, Burkitt's lymphoma, malignant lymphoma, diffuse lymphoma, andfollicular lymphoma), myeloma (including multiple myeloma), breastcancer, colon cancer, kidney cancer, gastric cancer, ovarian cancer,pancreatic cancer, cervical cancer, endometrial cancer, esophagealcancer, liver cancer, head and neck squamous cancer, skin cancer,urinary tract cancer, prostate cancer, choriocarcinoma, pharyngealcancer, laryngeal cancer, pleural tumor, arrhenoblastoma, endometrialhyperplasia, endometriosis, embryoma, fibrosarcoma, Kaposi's sarcoma,angioma, cavernous angioma, hemangioblastoma, retinoblastoma,spongiocytoma, neurofibroma, oligodendroglioma, medulloblastoma,neuroblastoma, neuroglioma, rhabdomyoblastoma, glioblastoma, osteogenicsarcoma, leiomyosarcoma, thyroid sarcoma, and Wilms tumor.
 92. Animmunosuppressive agent, which comprises, as an active ingredient, theantibody or a functional fragment thereof according to any one of claims30 to 40 and 77 to
 80. 93. A preventive, therapeutic, or diagnosticagent for autoimmune diseases or allergies, which comprises, as anactive ingredient, the antibody or a functional fragment thereofaccording to any one of claims 30 to 40 and 77 to 81.